TWI459963B - Pharmaceutical composition comprising an actriia-fc fusion protein; use of an actriia-fc fusion protein for treatment or prevention of cancer-related bone loss;use of an actriia-fc fusion protein for the treatment or prevention of multiple myeloma - Google Patents

Description

A pharmaceutical composition comprising an ActRIIa-Fc fusion protein; an use of an ActRIIa-Fc fusion protein for treating or preventing bone loss associated with cancer; an ActRIIa-Fc fusion protein for treatment or Prevention of multiple myeloma

The present invention relates to compositions and methods for promoting bone growth and increasing bone density as well as for treating multiple myeloma.

A bone condition ranging from osteoporosis to fracture represents a group of pathological conditions with few effective pharmaceutical agents. Treatment is instead focused on physical interventions and behavioral interventions, including fixation, exercise, and dietary changes. It would be beneficial to have a therapeutic agent that promotes bone growth and increases bone density for the purpose of treating a variety of bone disorders.

Bone growth and mineralization depend on the activity of the two cell types, osteoclasts and osteoblasts, although chondrocytes and vascular structural cells are also involved in key aspects of these processes. In terms of development, bone formation occurs through two mechanisms, intra-articular ossification and intramembranous ossification, the former responsible for longitudinal bone formation and the latter for topological flat bone (such as skull) formation. Endochondral ossification requires that cartilage structures form and degenerate sequentially in growth plates that serve as templates for osteoblasts, osteoclasts, vascular structure formation, and subsequent mineralization. During ossification of the membrane, bone is formed directly in connective tissue. Osteoblast infiltration and subsequent matrix deposition are required in both processes.

Fractures and other structural ruptures of the bone are restored by processes that are at least superficially similar to the sequence of developmental events of bone formation, including cartilage tissue formation and subsequent mineralization. The process of fracture recovery can occur in two ways. Direct or primary bone rejuvenation occurs without the formation of osteophytes. Indirect or secondary bone Recovery occurs with the epiphyseal stage of the epiphysis. Primary recovery of the fracture involves the formation of mechanical continuity across the high-density fracture. Under suitable conditions, bone resorbing cells around the rupture show a tunnel resorption response and establish a path for vascular penetration and subsequent recovery. Secondary recovery of bone follows the process of inflammation, cartilage formation, epiphyseal mineralization, and epiphyseal remodeling. During the inflammatory phase, hematoma and hemorrhage are caused by rupture of the periosteum and endosteal blood vessels at the injury site. Inflammatory cells invade this area. In the cartilage formation stage, the cells produce new blood vessels, fibroblasts, intracellular substances, and supporting cells, thereby forming granulation tissue in the space between the fracture fragments. The clinical healing system that spans the rupture is established by fibrous or cartilage tissue (cartilage). Osteoblasts are formed and mediate cartilage mineralization, which is then replaced by lamellar bone and undergoes a normal remodeling process.

In addition to fractures and other physical breakdown of bone structures, loss of bone mineral content and bone mass can be caused by a variety of conditions and can lead to significant medical problems. Changes in bone mass occur in a relatively predictable manner throughout an individual's lifetime. Until about 30 years old, the male and female bones grow to maximum mass through linear growth and radial growth of the endochondral growth plate. About 30 years old (for trabecular bones, such as flat bones, such as vertebrae and pelvis) and 40 years old (for cortical bones, such as long bones found in limbs), both men and women experience slow bone loss. In women, there is also a final stage of substantial bone loss, which may be due to a lack of estrogen after menopause. At this stage, women may lose an additional 10% bone mass from the cortical bone and an additional 25% bone mass from the trabecular region. Does progressive bone loss lead to pathological conditions such as osteoporosis that largely depend on the initial bone quality of the individual and Whether there is a worsening condition.

Bone loss is sometimes characterized by a dysregulation of the normal bone remodeling process. Healthy bones are often remodeled. Remodeling begins with bone resorption by osteoclasts. The resorbed bone is then replaced with new bone tissue characterized by collagen formation by osteoblasts followed by calcification. In healthy individuals, the rate of resorption is balanced with the rate of formation. Osteoporosis is a chronic, progressive condition characterized by a shift toward resorption, which results in a general decrease in bone mass and bone mineralization. Human osteoporosis precedes clinical bone loss (below the standard deviation of bone minerals below the standard deviation of young adults) by more than one standard deviation but below 2.5 standard deviations. Worldwide, approximately 75 million people are at risk of osteoporosis.

Thus, methods for controlling the balance between osteoclast activity and osteoblast activity can be applied to promote the recovery of fractures and other bone damage as well as the treatment of conditions associated with bone mass and loss of bone mineralization, such as osteoporosis. .

Regarding osteoporosis, estrogen, calcitonin, osteocalcin and vitamin K, or high doses of dietary calcium are used as therapeutic interventions. Other treatments for osteoporosis include bisphosphonates, parathyroid hormones, calciimetic, statins, anabolic steroids, guanidinium salts and guanidinium salts, and sodium fluoride. However, such therapeutic agents are often associated with undesirable side effects.

Accordingly, it is an object of the present invention to provide compositions and methods for promoting bone growth and mineralization.

Summary of invention

In part, the disclosure of this case demonstrates that molecules with activin or ActRIIa antagonist activity ("activin antagonists" and "ActRIIa antagonists", collectively referred to as "activin-ActRIIa antagonists") can be used to increase bone density and promote bone. Growing and / or increasing bone strength. In particular, the disclosure of this case demonstrates that the soluble form of ActRIIa acts as an inhibitor of activin-ActRIIa signaling and promotes increased bone density, bone growth and bone strength in vivo. Although most pharmaceutical agents that promote bone growth or inhibit bone loss act as anti-catabolic agents (also commonly referred to as "catabolic agents") (eg, bisphosphonates) or as anabolic agents (eg, The parathyroid hormone PTH, when properly administered), but the soluble ActRIIa protein exhibits dual activity, which is resistant to catabolism and assimilation. Thus, the disclosure of the present invention establishes that an antagonist of the activin-ActRIIa signal transduction pathway can be used to increase bone density and promote bone growth. Although soluble ActRIIa may affect bone by a different mechanism than activin antagonism, the disclosure herein confirms that the desired therapeutic agent can be selected based on activin-ActRIIa antagonist activity. Thus, in certain embodiments, the disclosure of the present disclosure provides for the use of, for example, activin-binding ActRIIa polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, targeting activins or small molecules targeting ActRIIa and aptamers (aptamer). Activin-ActRIIa antagonists, and nucleic acids that reduce activin and ActRIIa expression, treat conditions associated with low bone mineral density or low bone strength (such as osteoporosis) or promote patients in need (such as patients with fractures) ) The method of bone growth. The disclosure of this case further confirms activin-ActRIIa Antagonists are effective in preventing and/or repairing bone damage caused by multiple myeloma and breast tumors and additionally confirm that activin-ActRIIa antagonists reduce tumor burden in multiple myeloma. Soluble ActRIIa polypeptide promotes bone growth without causing consistent increases in muscle mass.

In certain aspects, the disclosure of the present invention provides a soluble activin-binding ActRIIa polypeptide comprising a binding to activin. The ActRIIa polypeptide can be formulated into a pharmaceutical preparation comprising an activin-binding ActRIIa polypeptide and a pharmaceutically acceptable carrier. Preferably, the activin-binding ActRIIa polypeptide of less than 1 micromolar or less than 100, 10 or K _D of 1 nemorubicin ear activin binding. Optionally, the activin-binding ActRIIa polypeptide with respect to GDF11 and / or GDF8 selectively binds activin, and preferably in a ratio of K _D to GDF11 and / or GDF8 of at least 10-fold, 20-fold or 50-fold and the K _D Activin binding. Although not wishing to be bound by a particular mechanism of action, it is expected that the selectivity of activin inhibition over GDF11/GDF8 inhibition is a factor that has a selective effect on bone and has no consistent measurable effect on muscle. In many embodiments, the ActRIIa polypeptide will be selected to cause a muscle increase of less than 15%, less than 10%, or less than 5% at a dose that achieves the desired effect on the bone. Preferably, the composition has a purity of at least 95% relative to the other polypeptide component as assessed by size exclusion chromatography, and more preferably, the composition has a purity of at least 98%. The activin-binding ActRIIa polypeptide used in the preparation may be any of the polypeptides disclosed herein, such as a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 7 or 12. Or a polypeptide having an amino acid sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence selected from SEQ ID NO: 2, 3, 7, 12 or 13. The activin-binding ActRIIa polypeptide may comprise a functional fragment of a native ActRIIa polypeptide, such as SEQ ID NO: 2 comprising a sequence selected from the group consisting of SEQ ID NO: 1-3 or lacking a C-terminal 10 to 15 amino acids ("tail"). A functional fragment of at least 10, 20 or 30 amino acids of the sequence.

A soluble activin-binding ActRIIa polypeptide can include one or more variations in an amino acid sequence (eg, a ligand binding domain) relative to a naturally occurring ActRIIa polypeptide. Examples of variant ActRIIa polypeptides are provided in WO 2006/012627, pages 59-60, which is incorporated herein by reference. Variations in the amino acid sequence can, for example, alter the glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or the protein cleavage of the altered polypeptide relative to the naturally occurring ActRIIa polypeptide.

The activin-binding ActRIIa polypeptide may be a fusion protein having an ActRIIa polypeptide (eg, a ligand binding portion of ActRIIa) as a domain and one or more additional domains providing desired properties, such as improved pharmacokinetics, Easy purification, targeting of specific tissues, etc. For example, the domain of the fusion protein may enhance one or more of in vivo stability, in vivo half-life, absorption/administration, tissue localization or distribution, protein complex formation, fusion protein multimerization, and/or purification. . Dimerization or multimerization can provide increased ligand binding affinity. The activin-binding ActRIIa fusion protein may comprise an immunoglobulin Fc domain (wild type or mutant) or serum albumin or other specific polypeptide moiety required to provide, for example, improved pharmacokinetics, improved solubility or improved stability. . Typically, the ActRIIa-Fc fusion protein will be produced as a homodimeric complex. Depending on the case, the ActRIIa-Fc fusion comprises a relatively unstructured linker between the Fc domain and the extracellular ActRIIa domain. The unstructured linker may correspond to about 15 amino acid unstructured regions ("tails") at the C-terminus of the extracellular domain of ActRIIa, or it may be 1, 2, 3, 4 Or an artificial sequence of 5 amino acids or between 5 and 15, 20, 30, 50 or more amino acids, an artificial sequence having no secondary structure, or both mixture. The linker may be rich in glycine and proline residues and may, for example, contain a single sequence of sulphonic acid/silicic acid and glycine or a repeat sequence of sulphonic acid/silicic acid and glycine ( For example, TG ₄ or SG ₄ single or repeat). Fusion proteins can include purified sequences, such as epitope tags, FLAG tags, polyhistidine sequences, and GST fusions. Optionally, the soluble ActRIIa polypeptide comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetamidine amines a base acid, an amino acid bound to biotin, an amino acid bound to a lipid moiety, and an amino acid combined with an organic derivatizing agent. The pharmaceutical preparations may also include one or more other compounds, such as compounds for the treatment of bone disorders. Preferably, the pharmaceutical preparation is substantially pyrogen free. In general, the ActRIIa protein is preferably expressed in a mammalian cell line that suitably mediates native glycosylation of the ActRIIa protein to reduce the likelihood of adverse immune responses in the patient. Human cell lines and CHO cell lines have been successfully used, and other commonly used mammalian expression systems are expected to be applicable.

As described herein, the ActRIIa protein (designated ActRIIa-Fc) has desirable properties, including selective binding to GDF8 and/or GDF11 to activin, high affinity ligand binding, and longer than two weeks in animal models. The serum half-life. In certain embodiments, the invention provides ActRIIa-Fc polypeptides and pharmaceutical formulations comprising the polypeptides and pharmaceutically acceptable excipients.

In certain aspects, the disclosure of the present invention provides a nucleic acid encoding a soluble activin-binding ActRIIa polypeptide. The isolated polynucleotide may comprise a coding sequence for a soluble activin-binding ActRIIa polypeptide, such as described above. For example, an isolated nucleic acid can include a sequence encoding an extracellular domain of ActRIIa (eg, a ligand binding domain) and will encode part or all of the transmembrane domain and/or cytoplasmic domain of ActRIIa, but encoding in a transmembrane domain or a cytoplasmic domain Sequence of stop codons either within or between the extracellular domain and the transmembrane domain or cytoplasmic domain. For example, an isolated polynucleotide can comprise a full-length ActRIIa polynucleotide sequence (such as SEQ ID NO: 4 or 5) or a partially truncated version, the isolated polynucleotide further comprising a transcription termination codon, which is At least six hundred nucleotides prior to the 3' terminus or otherwise positioned such that translation of the polynucleotide produces an extracellular domain that is fused to the truncated portion of full length ActRIIa as appropriate. A preferred nucleic acid sequence is SEQ ID NO: 14. The nucleic acids disclosed herein can be operably linked to a performance promoter, and the disclosure of the present disclosure provides cells transformed by such recombinant polynucleotides. Preferably, the cells are mammalian cells, such as CHO cells.

In certain aspects, the disclosure of the present invention provides methods of making a soluble activin-binding ActRIIa polypeptide. The method can include any of the nucleic acids disclosed herein (e.g., SEQ ID NO: 4, 5 or 14) in a suitable cell, such as a Chinese hamster ovary (CHO) cell. The method can comprise: a) culturing the cells under conditions suitable for the expression of a soluble ActRIIa polypeptide, Wherein the cell is transformed into a construct by soluble ActRIIa; and b) the soluble ActRIIa polypeptide thus expressed is recovered. The soluble ActRIIa polypeptide can be recovered as a crude, partially purified or highly purified fraction. Purification can be accomplished by a series of purification steps including, for example, one, two, or three or more of the following in any order: protein A chromatography, anion exchange chromatography (eg, Q agarose), hydrophobic interaction chromatography (eg phenyl sepharose), size exclusion chromatography and cation exchange chromatography.

In certain aspects, the activin-ActRIIa antagonists disclosed herein, such as soluble activin-binding ActRIIa polypeptides, can be used in methods that promote bone growth in an individual or increase bone density in an individual. In certain embodiments, the disclosure of the present invention provides a method of treating a patient in need thereof with a condition associated with low bone density or promoting bone growth in a patient in need thereof. The method can comprise administering an effective amount of an activin-ActRIIa antagonist to an individual in need thereof. In certain aspects, the disclosure of the present invention provides the use of an activin-ActRIIa antagonist for the manufacture of a medicament for treating a condition or condition as described herein.

In certain aspects, the disclosure of the present disclosure provides a method of identifying an agent that stimulates bone growth or stimulates increased bone mineralization. The method comprises: a) identifying a test agent that binds to a ligand binding domain of activin or an ActRIIa polypeptide; and b) assessing the effect of the agent on bone growth or bone mineralization.

Detailed description of the invention 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to have a biological effect on a variety of cell types in vertebrates and invertebrates. Members of the superfamily perform important functions in patterning and tissue specialization during embryonic development and can affect a variety of differentiation processes, including lipogenesis, muscle production, chondrogenesis, cardiac production, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two general branches: BMP/GDF and TGF-β/activin/BMP10 branches, the members of which have different, usually complementary roles. By manipulating the activity of members of the TGF-[beta] family, it is often possible to cause significant physiological changes in the organism. For example, the Piedmontese cattle and the Belgian Blue cattle breed carry a loss-of-function mutation in the GDF8 (also known as myostatin) gene, which causes a significant increase in muscle mass. Grobet et al., Nat Genet. 1997, 17(1): 71-4. Furthermore, in humans, the inactive allele of GDF8 is associated with increased muscle mass and is reported to be associated with abnormal intensity. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Activin is a dimeric polypeptide growth factor belonging to the TGF-β superfamily. There are three major activin forms (A, B, and AB), which are homodimers/heterodimers of two closely related beta subunits (β _A β _A , β _B β _{B ,} and β _A β _B ). The human genome also encodes activin C and activin E, which are mainly expressed in the liver. In the TGF-β superfamily, activin stimulates hormone production in ovarian cells and placental cells, supports neuronal cell survival, positively or negatively affects cell cycle progression depending on cell type, and induces mesoderm at least in amphibian embryos Unique and multifunctional factors of differentiation (DePaolo et al, 1991, Proc Soc Ep Biol Med. 198: 500-512; Dyson et al, 1997, Curr Biol. 7: 81-84; Woodruff, 1998, Biochem Pharmacol. 55: 953-963). Furthermore, red blood cell differentiation factor (EDF) isolated from stimulated human mononuclear leukemia cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85: 2434). Activin A has been shown to act as a natural positive regulator of red blood cell production in the bone marrow. In several tissues, activin signaling is antagonized by its associated heterodimeric statin. For example, during the release of follicle stimulating hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while statin prevents FSH secretion and synthesis. Other proteins that modulate activin biological activity and/or bind to activins include follistatin (FS), follistatin associated protein (FSRP), and alpha ₂ -macroglobulin.

The TGF-β signaling is mediated by a heteromeric complex of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream Smad proteins after ligand stimulation ( Massagu , 2000, Nat. Rev. Mol. Cell Biol. 1: 169-178). These Type I and Type II receptors are transmembrane proteins comprising a ligand-binding extracellular domain having a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted dextran/threonine specificity. Type I receptors are required for signal transduction; and type II receptors are required for binding to ligands and for expressing type I receptors. Type I and type II activin receptors form a stable complex upon ligand binding such that the type I receptor is phosphorylated by a type II receptor.

Two related type II receptors, ActRIIa and ActRIIb, have been identified as type II receptors for activins (Mathews and Vale, 1991, Cell 65: 973-982; Attisano et al, 1992, Cell 68: 97-108). In addition to activins, ActRIIa and ActRIIb interact biochemically with several other TGF-beta family proteins including BMP7, Nodal, GDF8 and GDF11 (Yamashita et al, 1995, J. Cell Biol. 130: 217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci. 98: 9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16: 2749-54). ALK4 is the major type I receptor for activins, particularly activin A, and ALK-7 can also act as a receptor for activins, particularly activin B.

As demonstrated herein, soluble ActRIIa polypeptide (sActRIIa), which shows substantial preferential binding to activin A in comparison to other TGF-beta family members (such as GDF8 or GDF11), is effective in promoting bone growth in vivo and increasing bone in vivo. density. Although not wishing to be bound by any particular mechanism, it is expected that the action of sActRIIa is primarily caused by the action of activin antagonists, assuming that the specific sActRIIa constructs used in these studies show very strong activin binding (Pymol dissociation constant) . Regardless of the mechanism, it is apparent from the data presented herein that ActRIIa-activin antagonists do increase bone density in normal mice, mouse models of osteoporosis, and mouse models of multiple myeloma. It should be noted that bone is a dynamic tissue whose growth or contraction and density increase or decrease the factors that cause bone and stimulate mineralization (mainly osteoblasts) and factors that destroy bone and demineralize it (mainly osteoclasts). ) depends on the balance. Bone growth and mineralization can be increased by increasing the production factor, by reducing the damage factor, or by both. The terms "promoting bone growth" and "increasing bone mineralization" refer to observable changes in bone entities and the mechanism for the occurrence of bone changes is intended to be neutral.

The mouse models of osteoporosis and bone growth/density used in the studies described herein are considered to be highly predictive of efficacy in humans, and thus, the disclosure of the present disclosure provides for the use of ActRIIa polypeptides and other activins - ActuIIa antagonists promote human bone growth and increase human bone density. Activin-ActRIIa antagonists include, for example, activin-binding soluble ActRIIa polypeptides, antibodies that bind to activins (especially activin A or B subunits, also known as βA or βB) and disrupt AotRIIa binding, bind to ActRIIa and disrupt Activin-binding antibodies, non-antibody proteins selected for activin or ActRIIa binding (for examples of such proteins and methods for designing and selecting such non-antibody affinity binding reagents, see, for example, WO/2002/088171, WO/ 2006/055689 and WO/2002/032925), and randomized peptides selected for activin or ActRIIa binding (usually attached to the Fc domain). Two different proteins (or other parts) with activin or ActRIIa binding activity, in particular blocking type I (eg soluble type I activin receptor) and type II (eg soluble type II activin receptor) binding sites, respectively The activin binding agents can be joined together to produce a bifunctional binding molecule. Nucleic acid aptamers, small molecules, and other agents that inhibit the activin-ActRIIa signal transduction axis can also be used. A variety of proteins have activin-ActRIIa antagonist activity, including statins (ie, statin alpha subunits) (although statins are not universally antagonizing activins in all tissues), follistatin (eg, follistatin) -288 and follistatin-315), FSRP, activin C, alpha(2)-macroglobulin, and M108A (methionine at position 108 becomes alanine) mutant activin A. In general, alternative forms of activin, especially those with mutations in the type I receptor binding domain, may Binding to a Type II receptor and failing to form an active ternary complex, thereby acting as an antagonist. In addition, a nucleic acid that inhibits activin A, B, C or E or particularly inhibits the expression of ActRIIa, such as an antisense molecule, siRNA or ribonuclease, can be used as an activin-ActRIIa antagonist. Preferably, the activin-ActRIIa antagonist to be used will show inhibition of activin-mediated signal transduction selectivity relative to GDF8 and GDF11, in contrast to other members of the TGF-[beta] family. The soluble ActRIIb protein does bind to activin. However, the wild-type protein does not show significant selectivity for binding of GDF8/11 to activin, and preliminary experiments have shown that this protein does not provide the desired effect on bone, but also causes substantial Muscle growth. However, variants of ActRIIb having different binding properties have been identified (see, for example, WO 2006/012627, pages 55-59, which is incorporated herein by reference), and such proteins can achieve the desired effect on bone. The native or variant ActRIIb can be administered by coupling with a second activin selective binding agent to increase the specificity for activin.

In the context of the present invention and in the particular case where various terms are used, the terms used in this specification generally have their ordinary meanings in the art. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of the terms will be apparent from the particular circumstances in which the term is used.

"Approximately" shall generally mean the degree of acceptable error in the amount measured under the nature or precision of the quantitative measurement. Typically, the degree of exemplary error is within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values.

Others and especially in biological systems, the term "about" may mean a value within one order of magnitude, preferably within 5 times and more preferably within 2 times of a given value. Unless otherwise stated, the quantities given herein are approximate, which means that the term "about" can be inferred when not explicitly stated.

The method of the invention can comprise the step of comparing sequences to each other, comprising comparing the wild type sequence to one or more mutants (sequence variants). Such comparisons typically involve aligning the polymer sequences, for example, using sequence alignment programs and/or algorithms well known in the art, such as BLAST, FASTA, and MEGALIGN, to name a few. It will be readily apparent to those skilled in the art that in such alignments, when a mutation contains a residue insertion or deletion, sequence alignment will introduce a "vacancy" in the polymer sequence that does not contain the inserted or deleted residue ( Typically represented by a dash or "A").

"Homologous" (all its grammatical forms and spelling changes) refers to the relationship between two proteins with "co-evolutionary origins", including proteins from superfamilies in the same organism species and from different organism species. a homologous protein. Such proteins (and their encoding nucleic acids) have sequence homology as reflected by their sequence similarity, based on percent identity or by the presence of specific residues or motifs and conserved positions.

The term "sequence similarity" (all grammatical forms thereof) refers to the degree of agreement or correspondence between nucleic acid sequences or amino acid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the application of the present invention, the term "homologous" may refer to sequence similarity when modified by an adverb such as "height" and may or may not involve a co-evolutionary origin.

2. ActRIIa polypeptide

In certain aspects, the invention relates to ActRIIa polypeptides. The term "ActRIIa" as used herein refers to a family of class IIa activin receptor (ActRIIa) proteins from any species and variants derived from such ActRIIa proteins by mutation or other modification. Reference to ActRIIa herein is to be understood as referring to any of the forms currently identified. Members of the ActRIIa family are typically transmembrane proteins comprising a ligand-binding extracellular domain rich in a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.

The term "ActRIIa polypeptide" includes polypeptides comprising any naturally occurring polypeptide of a member of the ActRIIa family and any variant (including mutants, fragments, fusions and peptidomimetics forms) that retain useful activity. For example, an ActRIIa polypeptide comprises any sequence of known ActRIIa derived from a sequence that is at least about 80% identical and preferably at least 85%, 90%, 95%, 97%, 99% or more identical to the sequence of the ActRIIa polypeptide. Peptide. For example, an ActRIIa polypeptide of the invention can bind to and inhibit the function of an ActRIIa protein and/or activin. Preferably, the ActRIIa polypeptide promotes bone growth and bone mineralization. Examples of ActRIIa polypeptides include human ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptide (eg, SEQ ID NOS: 2, 3, 7, and 12).

Human ActRIIa precursor protein sequence is as follows: MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRT N QTGVEPCYGDKDKRRHCFATWK N ISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNP VTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL (SEQ ID NO: 1)

The signal peptide is underlined; the extracellular domain is bold and the potential N-linked glycosylation site is double underlined.

The soluble (extracellular) processed polypeptide sequence of human ActRIIa is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCGNGNMCNEKFSYFPEM EVTQPISNPVTPKPP (SEQ ID NO: 2)

The C-terminal "tail" of the extracellular domain is underlined. The sequence of the "tail" deletion (Δ15 sequence) is as follows: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM (SEQ ID NO: 3)

The nucleic acid sequence encoding the human ActRIIa precursor protein is as follows (Genbank entry NM_001616 nucleotide 164-1705): ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATC TCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTG AGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTAT TAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA (SEQ ID NO: 4)

Encoding human ActRIIa soluble (extracellular) polypeptide is a nucleic acid sequence as follows: ATACTTGGTAGATCAGAAACTCAGGAGTGTC'TTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC (SEQ ID NO: 5)

In a specific embodiment, the invention relates to a soluble ActRIIa polypeptide. The term "soluble ActRIIa polypeptide" as used herein generally refers to a polypeptide comprising an extracellular domain of the ActRIIa protein. Terminology as used herein "Soluble ActRIIa polypeptide" includes any naturally occurring extracellular domain of the ActRIIa protein, as well as any variants thereof (including mutant, fragment and peptidomimetic forms). Activin-binding ActRIIa polypeptides are ActRIIa polypeptides that retain the ability to bind to activins, particularly activin AA, AB or BB. Preferably, the activin-binding ActRIIa polypeptide will bind to activin AA with a dissociation constant of 1 nM or less. The amino acid sequence of the human ActRIIa precursor protein is provided below. The extracellular domain of the ActRIIa protein binds to activin and is generally soluble, and thus may be referred to as a soluble activin-binding ActRIIa polypeptide. Examples of soluble activin-binding ActRIIa polypeptides include the soluble polypeptides set forth in SEQ ID NOS: 2, 3, 7, 12, and 13. SEQ ID NO: 7 is referred to as ActRIIa-hFc and is further described in the Examples. Other examples of soluble activin-binding ActRIIa polypeptides include signal sequences other than the extracellular domain of the ActRIIa protein, such as the honey melanotin leader sequence (SEQ ID NO: 8), tissue plasminogen activator (TPA). a leader (SEQ ID NO: 9) or a native ActRIIa leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptide described in SEQ ID NO: 13 uses a TPA leader.

A functionally active fragment of an ActRIIa polypeptide can be obtained by screening a polypeptide recombinantly produced from a corresponding fragment of a nucleic acid encoding an ActRIIa polypeptide. Alternatively, the fragments can be chemically synthesized using techniques known in the art, such as the conventional Merrifield solid phase f-Moc or t-Boc chemistry. Fragments can be generated (either recombinantly or by chemical synthesis) and tested to identify peptide-based fragments thereof that act as ActRIIa proteins or activin-mediated signal transduction antagonists (inhibitors).

A functionally active variant of an ActRIIa polypeptide can be obtained by screening a library of modified polypeptides recombinantly produced from the corresponding mutant nucleic acid encoding the ActRIIa polypeptide. Variants can be generated and tested to identify variants of antagonists (inhibitors) that can act as ActRIIa proteins or activin-mediated signal transduction. In certain embodiments, a functional variant of an ActRIIa polypeptide comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NO: 2 or 3. In certain instances, the functional variant has at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence selected from SEQ ID NO: 2 or 3. Amino acid sequence.

Functional variants can be produced by modifying the structure of the ActRIIa polypeptide for the purpose of enhancing therapeutic efficacy or stability, such as in vitro shelf life and resistance to proteolytic degradation in vivo. The modified ActRIIa polypeptides are considered to be functional equivalents of the naturally occurring ActRIIa polypeptide when selected to retain activin binding. The modified ActRIIa polypeptide can also be produced, for example, by substitution, deletion or addition of an amino acid. For example, it is reasonable to expect lysine to be separated or substituted with leucine or lysine, aspartate via glutamate, sulphite or serine, or amino acid via structure-related amine Similar substitutions of a base acid (e.g., conservative mutations) do not have a major effect on the biological activity of the resulting molecule. Conservative substitutions are those substitutions that occur in the family of amino acids that are related to their side chains. Whether a change in the amino acid sequence of an ActRIIa polypeptide produces a functional homolog can be readily determined by assessing the ability of the variant ActRIIa polypeptide to produce a response in a cell in a manner similar to the wild-type ActRIIa polypeptide.

In certain embodiments, the invention encompasses the specificity of an ActRIIa polypeptide Mutation to alter the glycosylation of the polypeptide. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. The aspartic acid-linked glycosylation recognition site generally comprises a tripeptide sequence aspartic acid-X-threonine (or aspartic acid-X-silk) specifically recognized by an appropriate cellular glycosylation enzyme. Amino acid) (wherein "X" is any amino acid). The variation may also be carried out by adding one or more serine or threonine residues to the sequence of the wild-type ActRIIa polypeptide or the sequence of the wild-type ActRIIa polypeptide via one or more serine or threonine residues. Substituted (for O-linked glycosylation sites). Substitution or deletion of a plurality of amino acids at one or both of the first or third amino acid positions of the glycosylation recognition site (and/or deletion of the amino acid at the second position) is modified Non-glycosylation occurs at the tripeptide sequence. Another way to increase the number of carbohydrate moieties on the ActRIIa polypeptide is by chemical or enzymatic coupling of the glycoside to the ActRIIa polypeptide. Depending on the coupling mode used, the sugar can be linked to: (a) arginine and histidine; (b) free carboxyl; (c) free sulfhydryl, such as cysteine a free sulfhydryl group; (d) a free hydroxyl group such as a free hydroxyl group of serine, threonine or hydroxyproline; (e) an aromatic residue such as phenylalanine, tyrosine or tryptophan Their aromatic residues; or (f) amidino groups of glutamic acid. Such methods are described in WO 87/05330, published Sep. 11, 1987, and by Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated herein by reference. in. Removal of one or more carbohydrate moieties present on the ActRIIa polypeptide can be accomplished chemically and/or enzymatically. Chemical deglycosylation can involve, for example, exposing an ActRIIa polypeptide to a compound trifluoromethanesulfonate Acid or equivalent compound. This treatment cleaves most or all of the sugar except the linking sugar (N-ethyl glucosamine or N-ethyl galactosamine) while leaving the complete amino acid sequence. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of the carbohydrate moiety on the ActRIIa polypeptide can be achieved by the use of various endoglycosidases and exoglycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350. When mammalian, yeast, insect, and plant cells can be introduced into different glycosylation patterns that can be affected by the amino acid sequence of the ActRIIa polypeptide, the sequence of the peptide can be adjusted as appropriate depending on the type of expression system used. Although other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are also contemplated, in general, the ActRIIa protein used in humans will be in a mammalian cell line that provides adequate glycosylation. Performance in (such as HEK293 or CHO cell lines).

The disclosure of the present invention further encompasses methods for producing mutants of ActRIIa polypeptides, particularly combinatorial mutants, and truncation mutants; combinatorial mutant sets are particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be to produce, for example, ActRIIa polypeptide variants that may act as agonists or antagonists or have novel activities in common. A variety of screening assays are provided below, and such assays can be used to evaluate variants. For example, an ActRIIa polypeptide variant can be screened for its ability to bind to an ActRIIa ligand, prevent the ActRIIa ligand from binding to an ActRIIa polypeptide, or interfere with signal transduction by an ActRIIa ligand.

Cell-based assays for the activity of ActRIIa polypeptides or variants thereof Or test in an in vivo assay. For example, the effect of an ActRIIa polypeptide variant on the performance of genes involved in bone production or bone destruction can be assessed. Where necessary, it can be carried out in the presence of one or more recombinant ActRIIa ligand proteins (e.g., activins), and the cells can be transfected to produce an ActRIIa polypeptide and/or variant thereof, and optionally an ActRIIa ligand. Likewise, an ActRIIa polypeptide can be administered to a mouse or other animal and one or more bone characteristics, such as density or volume, can be assessed. The rate of recovery of the fracture can also be assessed. The dual energy x-ray absorptiometry (DEXA) is a well established non-invasive quantitative technique for assessing bone density in animals. In humans, the central DEXA system can be used to assess bone density in the spine and pelvis. These densities are the best predictors of overall bone density. The peripheral DEXA system can be used to assess bone density in peripheral bone including, for example, the hand bone, the wrist bone, the tibia, and the foot bone. Traditional x-ray imaging systems including CAT scans can be used to assess bone growth and fracture recovery. The mechanical strength of the bone can also be assessed.

Combination derived variants that produce a selectivity or generally increased potency relative to a naturally occurring ActRIIa polypeptide can be produced. Likewise, mutations can produce variants having an intracellular half-life that is significantly different from the corresponding wild-type ActRIIa polypeptide. For example, a variant protein can be more stable or less stable to proteolytic degradation or other cellular processes that cause destruction or otherwise inactivation of the native ActRIIa polypeptide. Such variants and their coding genes can be used to alter the ActRIIa polypeptide content by modulating the half-life of the ActRIIa polypeptide. For example, short half-life can cause more transient biological effects and can allow for tighter control of recombinant ActRIIa polypeptide levels in a patient. In Fc fusion proteins, mutations can be made in the linker (if any) and/or Fc portion to change The half life of protein.

A combinatorial library can be produced via a gene degenerate library encoding a library of polypeptides each comprising at least a portion of a potential ActRIIa polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence such that a degenerate set of potential nucleotide sequences of the ActRIIa polypeptide can be expressed as an individual polypeptide or can be expressed as a set of larger fusion proteins (eg, For phage presentation).

There are many ways in which libraries of potential homologs can be generated from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, and the synthetic genes are then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland SymPos. Macromolecules, AG. Edited by Walton, Amsterdam: Elsevier, pp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been used in the directed evolution of other proteins (see, for example, Scott et al, (1990) Science 249: 386-390; Roberts et al, (1992) PNAS USA 89: 2429-2433; Devlin et al, ( 1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and U.S. Patent Nos. 5,223,409, 5,198,346 and 5,096,815.

Alternatively, other forms of mutations can be used to generate combinatorial libraries. For example, ActRIIa polypeptide variants can be generated and isolated from a library by screening using: for example, alanine scanning mutations and the like (Ruf Et al., (1994) Biochemistry 33: 1565-1572; Wang et al., (1994) J. Biol. Chem. 269: 3095-3099; Balint et al., (1993) Gene 137: 109-118; Grodberg et al. (1993) Eur. J. Biochem. 218: 597-601; Nagashima et al., (1993) J. Biol. Chem. 268: 2888-2892; Lowman et al., (1991) Biochemistry 30: 10832-10838; and Cunningham Et al., (1989) Science 244: 1081-1085); Linker Scanning Mutations (Gustin et al., (1993) Virology 193: 653-660; Brown et al., (1992) Mol. Cell Biol. 12: 2644-2652 ; McKnight et al, (1982) Science 232: 316); saturation mutations (Meyers et al, (1986) Science 232: 613); PCR mutations (Leung et al, (1989) Method Cell Mol Bioll: 11-19); Or random mutations (including chemical mutations, etc.) (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7: 32-34 ). Linker scanning mutations, especially in combination settings, are an attractive method for identifying truncated (biologically active) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening gene products of combinatorial libraries obtained by point mutations and truncation and, for this purpose, for screening for gene products having certain properties in cDNA libraries. Such techniques will generally be suitable for rapid screening of gene libraries generated by combinatorial mutations of ActRIIa polypeptides. The most widely used technique for screening large gene libraries typically involves the selection of a gene library into a replicable expression vector, transformation of the appropriate cells with the resulting vector library, and detection of the desired activity to facilitate coding of the gene (detection of its product) The vector exhibits a combinatorial gene under conditions in which the vector is relatively easily separated. Better check These include activin binding assays and activin-mediated cell signaling assays.

In certain embodiments, an ActRIIa polypeptide of the invention may further comprise a post-translational modification in addition to any of the naturally occurring ActRIIa polypeptides. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and deuteration. Thus, the modified ActRIIa polypeptide may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphate esters. The effect of such non-amino acid elements on the functionality of the ActRIIa polypeptide can be tested as described herein for other ActRIIa polypeptide variants. When an ActRIIa polypeptide is produced in a cell by cleavage of the nascent form of the ActRIIa polypeptide, post-translational processing is also important for proper folding and/or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure proper modification and processing of the ActRIIa polypeptide.

In certain aspects, a functional variant or modified form of an ActRIIa polypeptide comprises a fusion protein having at least a portion of an ActRIIa polypeptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistamine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin Protein heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin. The fusion domain can be selected to impart the desired characteristics. For example, some fusion domains are particularly useful for isolating fusion proteins by affinity chromatography. For affinity purification purposes, related matrices of affinity chromatography, such as glutathione binding resins, amylase binding resins, and nickel binding resins or cobalt binding resins, are used. Many of these matrices can "kit" form available, such as applicable to (HIS ₆₎ fusion Pharmacia GsT purification system and QIAexpress ^TM system with the object of (Qiagen). As another example, a fusion domain can be selected to facilitate detection of an ActRIIa polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "antigenic determinant tags", which are typically short peptide sequences available for a particular antibody. Well-known epitope tags that are readily available for specific monoclonal antibodies include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as Factor Xa or a protease cleavage site of thrombin, which allows the relevant protease to partially digest the fusion protein and thereby release the recombinant protein therefrom. The released protein can then be separated from the fusion domain by subsequent chromatographic separation. In certain preferred embodiments, the ActRIIa polypeptide is fused to a domain that stabilizes the ActRIIa polypeptide in vivo ("stabilizer" domain). "Stable" means any increase in serum half-life, whether due to reduced destruction, reduced renal clearance, or other pharmacokinetic effects. Fusion to the Fc portion of an immunoglobulin is known to confer a wide range of proteins with the desired pharmacokinetic properties. Likewise, fusion with human serum albumin can impart the desired properties. Other types of fusion domains that may be selected include poly (eg, dimeric, tetrameric) domains and functional domains (giving other biological functions, such as further stimulating bone growth or muscle growth if necessary).

As a specific example, the invention provides a fusion protein comprising a soluble extracellular domain of ActRIIa fused to the Fc domain (e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D (A) VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK (A ) VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSLSPGK *

Optionally, the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434. In certain instances, the ability of a mutant Fc domain having one or more of such mutations (eg, an Asp-265 mutation) to bind to an Fc gamma receptor relative to a wild-type Fc domain is reduced. In other instances, the ability of a mutant Fc domain having one or more of such mutations (eg, an Asn-434 mutation) to bind to a class I MHC-associated Fc receptor (FoRN) is increased relative to a wild-type Fc domain.

It should be understood that the different elements of the fusion protein can be arranged in any manner consistent with the desired functionality. For example, an ActRIIa polypeptide can be placed at the C-terminus of a heterologous domain or a heterologous domain can be placed at the C-terminus of an ActRIIa polypeptide. The ActRIIa polypeptide domain and the heterologous domain need not be adjacent in the fusion protein, and other domain or amino acid sequences may be included in or between the C-terminus or N-terminus of either domain.

In certain embodiments, an ActRIIa polypeptide of the invention contains one or more modifications that stabilize the ActRIIa polypeptide. For example, such modifications enhance the in vitro half-life of the ActRIIa polypeptide, enhance the circulating half-life of the ActRIIa polypeptide or reduce the proteolytic degradation of the ActRIIa polypeptide. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRIIa polypeptide and a stable subdomain), modifications of glycosylation sites (including, For example, a glycosylation site is added to the ActRIIa polypeptide), and a modification of the carbohydrate moiety (including, for example, removal of the carbohydrate moiety from the ActRIIa polypeptide). In the context of a fusion protein, the AotRIIa polypeptide is fused to a stable subdomain (such as the Fc domain) such as an IgG molecule. The term "stabilizing subdomain" as used herein refers not only to a fusion domain (eg, Fc) as in the case of a fusion protein, but also to non-proteinaceous modifications such as carbohydrate moieties or non-proteinaceous polymerizations such as polyethylene glycol. Things.

In certain embodiments, the invention produces an isolated and/or purified form of an ActRIIa polypeptide that is isolated or otherwise substantially free of other proteins from other proteins. AotRIIa polypeptides will generally be produced by expression from recombinant nucleic acids.

3. Nucleic acid encoding an ActRIIa polypeptide

In certain aspects, the invention provides isolated nucleic acids and/or recombinant nucleic acids encoding any of the ActRIIa polypeptides (eg, soluble ActRIIa polypeptides), including fragments, functional variants, and fusion proteins disclosed herein. For example, SEQ ID NO: 4 encodes a naturally occurring human ActRIIa precursor polypeptide, while SEQ ID NO: 5 encodes a processed ActRIIa extracellular domain. The subject nucleic acid can be single or double stranded. The nucleic acids can be DNA or RNA molecules. Such nucleic acids can be used, for example, in methods of making ActRIIa polypeptides or as direct therapeutic agents (eg, in gene therapy).

In certain aspects, a subject nucleic acid encoding an ActRIIa polypeptide is further understood to include a nucleic acid that is a variant of SEQ ID NO: 4 or 5. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.

In certain embodiments, the invention provides an isolated nucleic acid sequence or recombinant that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 5. Sex nucleic acid sequence. It will be understood by those skilled in the art that nucleic acid sequences complementary to variants of SEQ ID NO: 4 or 5 and SEQ ID NO: 4 or 5 are also within the scope of the invention. In other embodiments, the nucleic acid sequences of the invention may be isolated, recombinant, and/or fused to a heterologous nucleotide sequence, or in a DNA library.

In other specific embodiments, the nucleic acid of the present invention also includes a nucleoside which hybridizes under high stringency conditions to the nucleotide sequence represented by SEQ ID NO: 4 or 5, the complement of SEQ ID NO: 4 or 5, or a fragment thereof. Acid sequence. As discussed above, those of ordinary skill in the art will readily appreciate that the appropriate stringency conditions that promote DNA hybridization can be altered. Those of ordinary skill in the art will readily appreciate that the appropriate stringency conditions that promote DNA hybridization can be altered. For example, we can perform hybridization in 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C followed by washing with 2.0 x SSC at 50 °C. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x SSC at 50 °C. Additionally, the temperature in the washing step can be increased from low stringency conditions at room temperature (about 22 ° C) to high stringency conditions at about 65 ° C. Both temperature and salt can be varied, or the temperature or salt concentration can be kept constant while changing another variable. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by washing in 2 x SSC at room temperature.

An isolated nucleic acid that differs from the nucleic acid as set forth in SEQ ID NO: 4 or 5 due to degeneracy of the genetic code is also within the scope of the invention. For example Many amino acids are represented by more than one triplet. Designation of codons or synonymous codons of the same amino acid (e.g., CAU and CAC are synonymous codons for histidine) can cause "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that are not expected to cause changes in the amino acid sequence of the subject protein will be present in mammalian cells. Those skilled in the art will appreciate that such changes in one or more nucleotides (up to about 3-5% of the nucleotides) of a nucleic acid encoding a particular protein may be present in a particular species as a result of a natural allelic variation. Among the individuals. Any and all such nucleotide changes and resulting amino acid polymorphisms are within the scope of the invention.

In certain embodiments, a recombinant nucleic acid of the invention can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence will generally be suitable for the host cell for expression. A wide variety of suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells. Typically, the or such regulatory nucleotide sequence may include, but is not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, a transcriptional initiation and termination sequence, a translation initiation and termination sequence, And a enhancer sequence or an activator sequence. Constitutive or inducible promoters as known in the art are encompassed by the present invention. The promoter may be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct can be present on the clutching chromosome (such as a plastid) in the cell, or the expression construct can be inserted into the chromosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

In certain aspects of the invention, a subject nucleic acid is provided in a performance vector comprising a nucleotide sequence encoding an ActRIIa polypeptide and operably linked to at least one regulatory sequence. The regulatory sequences are technically recognized and selected to direct expression of the ActRIIa polypeptide. Thus, the term regulatory sequences include promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology : Methods in Enzymology , Academic Press, San Diego, CA (1990). For example, any of a variety of expression control sequences that control the expression of the DNA sequence when operably linked to a DNA sequence can be used in such vectors to express a DNA sequence encoding an ActRIIa polypeptide. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, adenovirus or cellular giant virus immediate early promoter, RSV promoter, lac system, trp system, TAC or TRC system, expression T7 promoter directed by T7 RNA polymerase, major operon and promoter region of phage lambda, control region of fd sheath protein, promoter of 3-phosphoglycerate kinase or other glycolytic enzyme, promoter of acid phosphatase (eg, Pho5), the promoter of the yeast alpha-mating factor, the polyhedrin promoter of the baculovirus system, and other sequences known to control the gene expression of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It will be appreciated that the design of the performance vector may depend on, for example, the choice of host cell to be transformed and/or the type of protein to be expressed. In addition, the number of copies of the vector, the ability to control the number of copies, and the performance of any other protein encoded by the vector (such as antibiotic markers) should also be considered.

The recombinant nucleic acid of the present invention can be produced by ligating the selected gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects or mammals) or both. . Expression vehicles that produce recombinant ActRIIa polypeptides include plastids and other vectors. For example, suitable vectors include plastids of the following types: plastids derived from pBR322 for expression in prokaryotic cells (such as E. coli ), plastids derived from pEMBL, derived from pEX The plastid, the plastid derived from pBTac and the plastid derived from pUC.

Some mammalian expression vectors contain prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are mammalian expression suitable for transfection of eukaryotic cells An example of a carrier. Some of these vectors are modified by sequences from bacterial plastids, such as pBR322, to facilitate replication and drug resistance selection in prokaryotic and eukaryotic cells. Alternatively, derivatives of the virus, such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP, and p205), can be used to transiently express proteins in eukaryotic cells. . Examples of other viral (including retroviral) expression systems can be found in the description of the gene therapy delivery system below. Various methods used in plastid preparation and in the transformation of host organisms are well known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, as well as general recombination procedures, see Molecular Cloning A Laboratory Manual , 3rd edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express a recombinant polypeptide by using a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393, and pVL941), vectors derived from pAcUW (such as pAcUW1), and vectors derived from pBlueBac (such as β-gal containing pBlueBac III). .

In a preferred embodiment, the vector will be designed to produce a subject ActRIIa polypeptide in a CHO cell, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), a pcDNA4 vector (Invitrogen, Carlsbad, Calif.). And pCI-neo vector (Promega, Madison, Wisc.). It will be apparent that the subject gene constructs can be used to facilitate expression of the subject ActRIIa polypeptide in cells that are propagated in culture, for example, to produce a protein comprising a fusion protein or a variant protein for purification.

The disclosure of the present invention is also directed to a host cell transfected with a recombinant gene comprising a coding sequence for one or more of the subject ActRIIa polypeptides (e.g., SEQ ID NO: 4 or 5). The host cell can be any prokaryotic or eukaryotic cell. For example, an ActRIIa polypeptide of the invention can be expressed in bacterial cells such as E. coli, insect cells (eg, using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the invention further relates to methods of producing the subject ActRIIa polypeptide. For example, a host cell transfected with an expression vector encoding an ActRIIa polypeptide can be cultured under appropriate conditions to allow expression of the ActRIIa polypeptide to occur. The ActRIIa polypeptide can be secreted and isolated from a mixture of cells containing the ActRIIa polypeptide and the culture medium. Alternatively, the ActRIIa polypeptide can be maintained in the cytoplasm or in the membrane fraction, and the cells are collected, solubilized and protein separated. quality. Cell cultures include host cells, culture media, and other by-products. Suitable media for cell culture are well known in the art. The subject ActRIIa polypeptide can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and use. An antibody specific for a particular epitope of an ActRIIa polypeptide is immunoaffinity purified and affinity purified using a reagent that binds to the domain fused to the ActRIIa polypeptide (eg, a Protein A column can be used to purify the ActRIIa-Fc fusion). In a preferred embodiment, the ActRIIa polypeptide is a fusion protein comprising a domain that facilitates its purification. In a preferred embodiment, the purification is achieved by a series of column chromatography steps comprising, for example, three or more of the following in any order: Protein A Chromatography, Q Sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be accomplished by viral filtration and buffer exchange. As demonstrated herein, the ActRIIa-hFc protein was purified to >98% as determined by size exclusion chromatography and >95% purity as determined by SDS PAGE. This purity is sufficient to achieve the desired effect on the bone of the mouse and to achieve acceptable safety features in mouse, rat and non-human primates.

In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly(His)/Enterokinase cleavage site sequence at the N-terminus of a desired portion of a recombinant ActRIIa polypeptide, may allow for the use of Ni ²⁺ metal by affinity chromatography. The resin is purified by the resin. The purified leader sequence can then be removed by treatment with enterokinase to provide a purified ActRIIa polypeptide (see, for example, Hochuli et al, (1987) J. Chromatography 411: 177; and Janknecht et al, PNAS USA 88: 8972 ).

Techniques for making fusion genes are well known. Essentially, the ligation of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques, using blunt ends or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling of sticky ends as appropriate, and avoidance of inconsistency The desired alkaline phosphatase treatment and enzymatic bonding are carried out. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of a gene fragment can be performed using anchoring primers that create complementary suspensions between two consecutive gene segments, which can then be ligated to produce a chimeric gene. Sequences (see, for example, Current Protocols in Molecular Biology , edited by Ausubel et al., John Wiley & Sons: 1992).

4. Replacement of Activin and ActRIIa Antagonists

The data presented herein demonstrate that antagonists of activin-ActRIIa signaling can be used to promote bone growth and bone mineralization. Although soluble ActRIIa polypeptides and especially ActrIIa-Fc are preferred antagonists and although such antagonists may be antagonized by different activins (eg, activin inhibition may be a molecular inhibition of agents that may include other members of the TGF-beta superfamily) The indicator of the trend of activity, and the mechanism by which this inhibition inhibits bone production, affects bone, but other types of activin-ActRIIa antagonists are expected to be applicable, including anti-activins (eg A, B, C or E) antibodies, anti-ActRIIa antibodies, antisense RNAi or ribonuclease nucleic acids that inhibit ActRIIa production, and activins or other inhibitors of ActRIIa, particularly which inhibit the binding of activin-ActRIIa.

An antibody that specifically reacts with an ActRIIa polypeptide (eg, a soluble ActRIIa polypeptide) and that competitively binds to an ActRIIa polypeptide or otherwise inhibits ActRIIa-mediated signal transduction can be used as an antagonist of ActRIIa polypeptide activity. Similarly, an antibody that specifically reacts with the activin A polypeptide and disrupts the binding of ActRIIa can be used as an antagonist.

Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols by using immunogens derived from ActRIIa polypeptides or activin polypeptides (see, for example, Antibodies: A Laboratory Manual by Harlow and Lane (Cold Spring Harbor Press) :1988)). Mammals such as mice, hamsters or rabbits can be immunized with an immunogenic form of an ActRIIa polypeptide, an antigenic fragment capable of eliciting an antibody response, or a fusion protein. Techniques for imparting immunogenicity to a protein or peptide include binding to a carrier or other techniques well known in the art. The immunogenic portion of ActRIIa or activin polypeptide can be administered in the presence of an adjuvant. The process of immunization can be monitored by detecting antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as an antigen to assess antibody content.

After immunizing the animal with the antigen preparation of the ActRIIa polypeptide, antiserum can be obtained and, if necessary, multiple antibodies can be isolated from the serum. To produce a monoclonal antibody, antibody-producing cells (lymphocytes) can be collected from the immunized animal and fused with immortalized cells (such as myeloma cells) by standard somatic cell fusion procedures to obtain fusion tumor cells. Such techniques are well known in the art and include, for example, fusion tumor technology (originally developed by Kohler and Milstein, (1975) Nature, 256:495-497), human B cell fusion tumor technology (Kozbar et al. , (1983) Immunology Today, 4:72) EBV-fused tumor technology of human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). The fusion tumor cells can be screened immunochemically to produce antibodies that specifically react with the ActRIIa polypeptide and monoclonal antibodies isolated from cultures comprising such fusion tumor cells.

The term "antibody" as used herein is intended to include fragments thereof which also specifically react with a subject polypeptide. Antibodies can be fragmented using conventional techniques and screened for utility in the same manner as described above for the entire antibody. For example, a F(ab) ₂ fragment can be produced by treating an antibody with pepsin. The resulting F(ab) ₂ fragment can be treated to reduce the disulfide bridge to produce a Fab fragment. The antibodies of the invention are further intended to include bispecific, single chain, chimeric, humanized and fully human molecules having an affinity for an ActRIIa or activin polypeptide conferred by at least one CDR region of the antibody. The antibody may further comprise a label attached thereto and detectable (eg, the label may be a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor).

In certain embodiments, the antibody is a recombinant antibody, the term encompasses any antibody produced in part by techniques of molecular biology, including CDR-grafted or chimeric antibodies, human antibodies assembled from antibody-selected antibody domains, or Other antibodies, single chain antibodies, and single domain antibodies (eg, human _VH protein or camel _VHH protein). In certain embodiments, the antibodies of the invention are monoclonal antibodies, and in certain embodiments, the invention provides a method of producing novel antibodies. For example, a method of producing a monoclonal antibody that specifically binds to an ActRIIa polypeptide or an activin polypeptide can comprise administering to a mouse an amount of an immunogenic composition comprising an antigenic polypeptide that is effective to stimulate an immunoreactive response. The mouse obtains antibody-producing cells (for example, cells derived from the spleen) and fuses the antibody-producing cells with myeloma cells to obtain antibody-producing fusion tumors, and tests the antibodies to produce fusion tumors to identify specific binding to antigens. A fusion tumor of a single antibody. Once obtained, the fusion tumor can be propagated in cell culture, optionally under conditions of culture of monoclonal antibodies that specifically bind to the antigen from cells derived from the fusion tumor. Individual antibodies can be purified from cell culture.

As used in relation to antibodies, the adjective "specifically reacts with" is intended to mean that antibodies are generally known to have sufficient selectivity between an antigen of interest (e.g., an ActRIIa polypeptide) and other non-antibiotics of interest. The antibody is suitable for detecting the presence of an antigen of interest in a particular type of biological sample at a minimum. In certain methods of using antibodies, such as therapeutic applications, a higher degree of binding specificity may be desirable. Individual antibodies generally have a large tendency to effectively distinguish between the desired antigen and the cross-reactive polypeptide (compared to multiple antibodies). One characteristic that affects the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen. While the desired specificity can be achieved with a range of different affinities, generally preferred antibodies will have an affinity (dissociation constant) of about 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 or less. Given that the binding between activin and ActRIIa is extremely tight, it is expected that neutralizing anti-activin or anti-ActRIIa antibodies will generally have a dissociation constant of 10 ^-10 or less.

In addition, techniques for screening antibodies to identify desired antibodies can affect the properties of the antibodies obtained. For example, if an antibody is to be used to bind an antigen in a solution, then test solution binding may be required. A variety of different techniques can be used to test the interaction between an antibody and an antigen to identify antibodies that are particularly desirable. Such techniques include ELISA, surface plasmon resonance binding assays (e.g. the Biacore ^(TM) binding assay, Biacore AB, Uppsala, Sweden) , sandwich assays (e.g. IGEN International, Inc., Gaithersburg, Maryland system of paramagnetic beads), Western blots Point method, immunoprecipitation assay and immunohistochemistry.

Examples of classes of nucleic acid compounds that act as activins or ActRIIa antagonists include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. The nucleic acid compound can be single or double stranded. The double-stranded compound may also include a pendant or non-complementary region, wherein one or the other of the strands is a single strand. A single-stranded compound may include a self-complementary region, which means that the compound forms a so-called "hairpin" or "stem loop" structure having a double helix structure. The nucleic acid compound may comprise no more than 1000, no more than 500, no more than 250, no more than 100 or no more than 50, 35, 30 of the nucleic acid sequence of the full-length ActRIIa nucleic acid sequence or activin βA or activin βB. A nucleotide sequence complementary to a region of 25, 22, 20 or 18 nucleotides. The complementary region will preferably be at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, and optionally between 15 nucleotides and 25 nucleotides. The complementary region may belong to an intron, a coding sequence or a non-coding sequence of a target transcript, such as a portion of a coding sequence. In general, the nucleic acid compound will have a length of from about 8 to about 500 nucleotides or base pairs in length, and will optionally be from about 14 to about 50 nucleotides in length. The nucleic acid can be DNA (especially used as antisense DNA), RNA or RNA:DNA hybrids. Any strand may include a mixture of DNA and RNA, as well as a modified form that cannot be readily classified as DNA or RNA. Similarly, double-strand The compound may be DNA: DNA, DNA: RNA or RNA: RNA, and any strand may also include a mixture of DNA and RNA and a modified form that cannot be easily classified as DNA or RNA. The nucleic acid compound can include any of a variety of modifications including one of a backbone (a sugar-phosphate moiety in a natural nucleic acid, including an internucleotide linkage) or a base moiety (a purine or pyrimidine moiety of a natural nucleic acid) or A variety of modifications. The antisense nucleic acid compound will preferably have a length of from about 15 to about 30 nucleotides and will typically contain one or more modifications to improve the characteristics such as in serum, in cells, or where the compound may be delivered (such as Stability in the stomach and in the lungs for inhaled compounds in the case of orally administered compounds. In the context of an RNAi construct, the strand complementary to the target transcript will generally be RNA or a modified form thereof. The other can be RNA, DNA or any other form of variation. The duplex portion of the double-stranded or single-stranded "hairpin" RNAi construct will preferably have a length of from 18 to 40 nucleotides in length and optionally from about 21 to 23 nucleotides in length, as long as it It can be used as a Dicer. The catalytic or enzymatic nucleic acid can be a ribonuclease or DNase and can also contain a modified form. The nucleic acid compound can inhibit the performance of the target by about 50%, 75%, 90% or more when it is contacted with the cells under physiological conditions and at a concentration with little or no effect in a nonsense or sense control. Preferred concentrations of the test nucleic acid compound are 1, 5 and 10 micromolar. Nucleic acid compounds can also be tested for effects on, for example, bone growth and mineralization.

5. Screening verification

In certain aspects, the invention relates to the identification of an ActRIIa polypeptide (eg, a soluble ActRIIa polypeptide) and an activin polypeptide as activin-ActRIIa Use of a compound (agent) of an agonist or antagonist of a signal transduction pathway. Compounds identified by this screen can be tested to assess their ability to modulate bone growth or mineralization in vitro. These compounds can be further tested in animal models to assess their ability to modulate tissue growth in vivo, as appropriate.

There are numerous methods for screening therapeutic agents that modulate tissue growth by targeting activin and ActRIIa polypeptides. In certain embodiments, high yield screening of compounds can be performed to identify agents that dampen activin or ActRIIa mediated effects on bone. In certain embodiments, assays are performed to screen for and identify compounds that specifically inhibit or reduce binding of the ActRIIa polypeptide to activin. Alternatively, assays can be used to identify compounds that enhance the binding of an ActRIIa polypeptide to activin. In yet another embodiment, the compounds can be identified by their ability to interact with activin or ActRIIa polypeptides.

A variety of verification patterns will suffice, and according to the disclosure of this case, the verification patterns not explicitly described herein are still known to those familiar with the art. As described herein, the test compounds (agents) of the invention can be produced by any combinatorial chemistry. Alternatively, the subject compound can be a naturally occurring biomolecule synthesized in vivo or in vitro. Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (eg, natural products), chemically produced (eg, small molecules, including peptide mimics) ()), or produced recombinantly. Test compounds encompassed by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In a particular embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons.

The test compounds of the invention may be provided in a single discrete entity form or in a library of higher complexity, such as a library made by combinatorial chemistry. Such libraries may contain, for example, alcohols, alkyl halides, amines, guanamines, esters, aldehydes, ethers, and other types of organic compounds. The presentation of the test compound to the test system can be in isolated form or as a mixture of compounds (especially in the initial screening step). The compound may optionally be derivatized with other compounds and have a derivatizing group that facilitates separation of the compound, as desired. Non-limiting examples of derivatizing groups include biotin, luciferin, digoxygenin, green fluorescent protein, isotope, polyhistamine, magnetic beads, glutathione S transferase (GST), Photoactivatable crosslinkers or any combination thereof.

In many drug screening programs that test libraries of compounds and natural extracts, high yield assays are required to maximize the number of compounds investigated over a given time period. Assays performed in cell-free systems such as those that can be purified or semi-purified are generally preferred as "primary" screens because such assays can be generated to allow rapid visualization and relatively easy detection of test compounds Mediated variation of molecular targets. In addition, the effect of the cytotoxicity or bioavailability of the test compound is generally neglected in an in vitro system, and the assay is primarily focused on the effect of the drug on the molecular target, which can be a variation in the binding affinity between the ActRIIa polypeptide and the activin. Shown in the middle.

For purposes of illustration only, in an exemplary screening assay of the invention, the compound of interest is contacted with an isolated and purified ActRIIa polypeptide that is normally capable of binding to activin. The composition containing the ActRIIa ligand is then added to the mixture of the compound and the ActRIIa polypeptide. ActRIIa/activin complex Detection and quantification of the compounds provides a means to determine the efficacy of a compound to inhibit (or potentiate) complex formation between the ActRIIa polypeptide and activin. The efficacy of a compound can be assessed by generating a dose response curve from data obtained using test compounds at multiple concentrations. In addition, a control check can be performed to provide a baseline for comparison. For example, in a control assay, the isolated and purified activin is added to a composition containing an ActRIIa polypeptide and the formation of the ActRIIa/activin complex is quantified in the absence of the test compound. It will be appreciated that, in general, the order in which the reactants are mixed may vary and may be mixed simultaneously. In addition, instead of purified proteins, cell extracts and lysates can be used to facilitate a suitable cell-free assay system.

Complex formation between ActRIIa polypeptides and activins can be detected by a variety of techniques. For example, modulation of complex formation can be used, for example, such as radiolabeled (eg, ³² P, ³⁵ S, ¹⁴ C, or ³ H), fluorescently labeled (eg, FITC) or enzymatically labeled ActRIIa polypeptides or Activin is a detectable marker protein that is quantified by immunoassay or by chromatographic detection.

In certain embodiments, the invention encompasses the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays to directly or indirectly measure the degree of interaction between an ActRIIa polypeptide and its binding protein. In addition, other detection modes, such as those based on optical waveguides (PCT Publication No. WO 96/26432 and U.S. Patent No. 5,677,196), surface plasma resonance (SPR), surface charge sensors, and surface force sensors The test mode is compatible with many specific examples of the invention.

In addition, the present invention covers interaction trap verification (also known as "double miscellaneous" The use of the assay, which is used to identify agents that disrupt or enhance the interaction between an ActRIIa polypeptide and its binding protein. See, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; And Iwabuchi et al. (1993) Oncogene 8: 1693-1696. In a specific embodiment, the invention encompasses the use of a reverse two-hybrid system to identify a compound (eg, a small molecule or peptide) that dissociates the interaction between an ActRIIa polypeptide and its binding protein. See, for example, Vidal and Legrain, (1999) Nucleic Acids Res 27: 919-29; Vidal and Legrain, (1999) Trends Biotechnol 17: 374-81; and U.S. Patent Nos. 5,525,490, 5,955,280 and 5,965,368.

In certain embodiments, the subject compounds are identified by their ability to interact with an ActRIIa or activin polypeptide of the invention. The interaction between a compound and an ActRIIa or activin polypeptide can be covalent or non-covalent. For example, the interaction can be identified at the protein level using in vitro biochemical methods including photocrosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46). :1). In certain instances, the compound can be screened in a mechanism-based assay, such as a assay to detect a compound that binds to activin or an ActRIIa polypeptide. It can include solid phase or liquid phase binding events. Alternatively, a gene encoding an activin or an ActRIIa polypeptide can be transfected into a cell via a reporter system (eg, β-galactosidase, luciferase, or green fluorescent protein) and is preferably screened for high-yield against the library or The library Individual members are screened. Other mechanism-based binding assays can be used, such as a combined assay that detects changes in free energy. Binding assays can be performed using targets immobilized on wells, beads or wafers or captured by immobilized antibodies or resolved by capillary electrophoresis. Colorimetric or fluorescent methods or surface plasma resonances can generally be used to detect the bound compound.

In certain aspects, the invention provides methods and medicaments for modulating (stimulating or inhibiting) bone formation and increasing bone mass. Thus, any compound identified can be tested in vitro or in vivo throughout the cell or tissue to confirm its ability to modulate bone growth or mineralization. A variety of methods known in the art can be used for this purpose.

For example, the effect of ActRIIa or activin polypeptide or test compound on bone or cartilage growth can be determined by measuring Msx2 induction or differentiation of osteoprogenitor cells into osteoblasts in a cell-based assay (see, for example, Daluiski et al. Human, Nat Genet. 2001, 27(1): 84-8; Hino et al., Front Biosci. 2004, 9: 1520-9). Another example of a cell-based assay includes analysis of the osteogenic activity of a subject ActRIIa or activin polypeptide and a test compound in mesenchymal progenitor cells and osteoblasts. For the sake of illustration, recombinant adenovirus expressing activin or ActRIIa polypeptide can be constructed to infect differentiated pluripotent mesenchymal progenitor cells C3H10T1/2 cells, pre-osteogenic C2C12 cells, and osteoblast TE-85 cells. Osteogenic activity is then determined by measuring the induction of alkaline phosphatase, osteocalcin and matrix mineralization (see, for example, Cheng et al, J bone Joint Surg Am. 2003, 85-A(8):1544-52 ).

The invention also encompasses in vivo assays for measuring bone or cartilage growth. For example, Namkung-Matthai et al., Bone, 28: 80-86 (2001) reveals A rat osteoporosis model for early bone repair after fracture was studied. Kubo et al, Steroid Biochemistry & Molecular Biology, 68: 197-202 (1999) also revealed a rat osteoporosis model for the study of late bone repair after fracture. Andersson et al., J. Endocrinol. 170:529-537 describe a mouse model of osteoporosis in mice undergoing ovariectomy. Ovariectomy results in loss of substantial bone mineral content and bone mineral density in these mice, with trabecular bone loss. About 50% of bone mineral density. The bone density of the ovariectomized mouse can be increased by administering a factor such as parathyroid hormone. In certain aspects, the present invention uses a fracture recovery assay known in the art. Such assays include fracture techniques, histological analysis, and biomechanical analysis, which are described, for example, in U.S. Patent No. 6,521,750, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in The way to merge.

6. Exemplary therapeutic uses

In certain embodiments, an activin-ActRIIa antagonist (eg, an ActRIIa polypeptide) of the invention can be used to treat or prevent a disease or condition associated with, for example, bone damage caused by rupture, loss, or demineralization. As demonstrated herein, activin-ActRIIa antagonists, and in particular ActRIIa-Fc constructs, are effective in treating or preventing cancer-related bone loss. In certain embodiments, the invention provides methods of treating or preventing bone damage in a subject by administering a therapeutically effective amount of an activin-ActRIIa antagonist, particularly an ActRIIa polypeptide, to an individual in need thereof. In certain embodiments, the invention provides a method for promoting bone growth or mineralization of a subject by administering a therapeutically effective amount of an activin-ActRIIa antagonist, particularly an ActRIIa polypeptide, to an individual in need thereof. law. These methods are preferably directed to the therapeutic and prophylactic treatment of animals and better humans. In certain embodiments, the disclosure of the present invention provides activin-ActRIIa antagonists (particularly soluble ActRIIa polypeptides and targeted activins or ActRIIa neutralizing antibodies) for the treatment of conditions associated with low bone density or reduced bone strength. use.

As used herein, a "prevention" of a condition or condition is a decrease in the incidence of a condition or condition of a treated sample relative to an untreated control sample or a delay relative to an untreated control sample in a statistical sample. A compound that causes the onset of one or more symptoms of a condition or condition or reduces the severity of one or more symptoms of the condition or condition. The term "treating" as used herein includes the prevention of a specified condition or once the condition has indeed improved or eliminated the condition. In either case, the prophylaxis or treatment can be discerned in the diagnosis provided by the physician and the desired result of administering the therapeutic agent.

The disclosure of the present invention provides a method of inducing bone and/or cartilage formation, preventing bone loss, increasing bone mineralization, or preventing bone demineralization. For example, the subject activin-ActRIIa antagonist has applications for treating osteoporosis in humans and other animals and restoring fractures and cartilage defects in humans and other animals. ActRIIa or activin polypeptides may be useful as protective measures for the development of osteoporosis in patients diagnosed with subclinical low bone density.

In a particular embodiment, the methods and compositions of the present invention have medical utility in restoring fractures and cartilage defects in humans and other animals. The subject methods and compositions can also have prophylactic uses in terms of occlusion and open fracture reduction as well as improved fixation of artificial joints. Re-bone formation induced by osteogenic agents contributes to congenital, traumatic or tumor resection The repair of induced craniofacial defects is also applicable to plastic surgery. In certain instances, the subject activin-ActRIIa antagonist can provide an environment that attracts bone-forming cells, stimulates bone-forming cell growth, or induces differentiation of progenitor cells of bone-forming cells. The activin-ActRIIa antagonist of the present invention can also be used for the treatment of osteoporosis.

The methods and compositions of the present invention are applicable to conditions characterized by loss of bone or bone loss, such as osteoporosis (including secondary osteoporosis), parathyroid dysfunction, Cushing's disease ( Cushing's disease), Paget's disease, thyrotoxicosis, chronic diarrhea or malabsorption, renal tubular acidosis or anorexia nervosa.

Osteoporosis can be caused by a variety of factors or associated with a variety of factors. As a woman, especially after menopause, light weight and sedentary lifestyle are risk factors for osteoporosis (loss of bone mineral density, leading to fracture risk). A person having any of the following characteristics may be a candidate for treatment with an ActRIIa antagonist: a post-menopausal woman who has not taken estrogen or other hormone replacement therapy; a person with a hip fracture or smoking personal or maternal history; high (more than 5 suction 7 inches) or thin body (less than 125 pounds) of the menopause in women after; masculine correlation of clinical symptoms and bone loss of; known to cause drug loss of bone (including corticosteroids such as Prednisone ^TM) , various anti-burst drugs (such as Dilantin ^TM) and certain barbiturates (barbiturate) or high-dose thyroid replacement drugs of the person; type 1 diabetes, liver disease, kidney disease or a family history of osteoporosis, having a person; having People with high bone turnover (eg, excess collagen in urine); people with thyroid conditions (such as thyroid hyperactivity); people who suffer fractures only after minor trauma; x-ray signs of vertebral fractures or osteoporosis Other signs of people.

As mentioned above, osteoporosis can also be caused as a condition associated with another condition or by the use of certain drugs. Osteoporosis caused by drugs or another medical condition is called secondary osteoporosis. In a condition known as Cushing's disease, excess cortisol produced by the body causes osteoporosis and fracture. The most common drug associated with secondary osteoporosis is corticosteroids, a class of drugs that act like cortisol, a hormone naturally produced by the adrenal glands. Although a sufficient amount of thyroid hormone (which is produced by the thyroid gland) is required for skeletal development, excessive thyroid hormone can reduce bone mass over time. Aluminum-containing antacids can cause bone loss when taken at high doses by people with kidney problems, especially those who have undergone dialysis. Other drugs that can cause secondary osteoporosis include: phenytoin (Dilantin) and barbiturate, which are used to prevent bursts; methotrexate (Rheumatrex, Immunex, Folex PFS), a a drug for some forms of arthritis, cancer, and immune disorders; cyclosporine (Sandimmune, Neoral), a drug used to treat some autoimmune diseases and suppress the immune system of organ transplant patients; Hormone-releasing hormone agonist (Lupron, Zoladex) for the treatment of prostate cancer and endometriosis; heparin (Calciparine, Liquaemin), an anticoagulant; and cholestyramine (Questran) Colestipol (Colestid), which is used to treat high cholesterol. Bone loss caused by cancer therapy is common Understand and call cancer therapy-induced bone loss (CTIBL). Bone metastases can create cavities in the bone that can be corrected by treatment with activin-ActRIIa antagonists.

In a preferred embodiment, the activin-ActRIIa antagonists disclosed herein, particularly soluble ActRIIa, are useful in cancer patients. Patients with certain tumors (eg, prostate tumors, breast tumors, multiple myeloma, or any tumor that causes parathyroid gland hyperplasia) are at high risk of tumor-induced bone loss and bone metastasis and bone loss caused by therapeutic agents . These patients can be treated with activin-ActRIIa antagonists even in the absence of signs of bone loss or bone metastasis. Patients may also be monitored for signs of bone loss or bone metastasis, and such patients may be treated with an activin-ActRIIa antagonist if the indicator indicates an increased risk. In general, DEXA scans are used to assess changes in bone density, and indicators of bone remodeling can be used to assess the likelihood of bone metastasis. Serum markers can be monitored. Bone-specific alkaline phosphatase (BSAP) is an enzyme found in osteoblasts. The blood content of BSAP is increased in patients with bone metastases and other conditions that result in increased bone remodeling. Osteocalcin and procollagen peptides are also associated with bone formation and bone metastasis. BSAP increase has been detected in patients with bone metastases caused by prostate cancer and to a lesser extent in bone metastases from breast cancer. Bone morphogenetic protein-7 (BMP-7) levels are higher in prostate cancer that has metastasized to bone, but not in bone metastases caused by bladder, skin, liver or lung cancer. Type I carboxy terminal tail peptide (ICTP) is a crosslinker found in collagen formed during bone resorption. Because bones often rupture and re-form, ICTP is found throughout the body. However, in the area of bone metastasis The content will be significantly higher than the area of normal bone. High levels of ICTP have been found in bone metastases caused by prostate cancer, lung cancer, and breast cancer. Another collagen cross-linker type I N-terminal peptide (NTx) is produced along with ICTP during bone turnover. The amount of NTx is increased in bone metastases caused by many different types of cancer including lung cancer, prostate cancer, and breast cancer. Also, the amount of NTx increases with the 'process of bone transfer'. Therefore, this marker can be used to detect metastases and measure disease levels. Other markers of resorption include pyridinoline and deoxypyridinoline. Any increase in the reabsorption marker or bone metastasis marker is indicative of the patient's need for activin-ActRIIa antagonist therapy.

Activin-ActRIIa antagonists can be administered in combination with other pharmaceutical agents. Combination administration can be achieved by administering a single co-administered formulation, by administering the drug simultaneously, or by administering the drug at separate times. Activin-ActRIIa antagonists are especially advantageous when administered with other bone active agents. Patients may benefit from a combination of an activin-ActRIIa antagonist and a calcium supplement, vitamin D, proper exercise, and/or other medications under some conditions. Examples of other drugs include bisphosphonates (alendronate, ibandronate and risedronate), calcitonin, estrogen, parathyroid hormone and Raloxifene. Bisphosphonates (alendronate, ibandronate, and risedronate), calcitonin, estrogen, and raloxifene affect the bone remodeling cycle and are classified as anti-resorptive drugs. Bone remodeling consists of two distinct phases: bone resorption and bone formation. The anti-resorptive drug slows or stops the bone resorption portion of the bone remodeling cycle, but does not slow the bone formation portion of the cycle. Therefore, the new formation is faster than bone resorption Continue, and bone density can increase over time. One form of parathyroid hormone, teriparatide, increases the rate of bone formation during the bone remodeling cycle. Alendronate is approved for the prevention of postmenopausal osteoporosis (5 mg daily or 35 mg once a week) and treatment (10 mg per day or 70 mg once a week). Alendronate reduces bone loss, increases bone density and reduces the risk of spinal, wrist and hip fractures. Alendronate is also approved for the treatment of glucocorticoid-induced osteoporosis in men and women due to long-term use of glucocorticoids (ie, prednisone and cortisone) For the treatment of osteoporosis in men. Alendronate plus vitamin D is approved for the treatment of osteoporosis in postmenopausal women (plus 70 mg once a week for vitamin D) and is used to improve the bone quality of men with osteoporosis. Ibandronate is approved for the prevention and treatment of postmenopausal osteoporosis. Take it once a month in pill (150 mg) and ibandronate should be taken on the same day of the month. Ibandronate reduces bone loss, increases bone density and reduces the risk of spinal fractures. Risedronate is approved for the prevention and treatment of postmenopausal osteoporosis. Taking it daily (5 mg dose) or weekly (35 mg dose or 35 mg dose with calcium), risedronate slows bone loss, increases bone density and reduces the risk of spinal fractures and non-spine fractures. Risedronate has also been approved for use by men and women to prevent and/or treat glucocorticoid-induced osteoporosis caused by long-term use of glucocorticoid drugs (ie, prednisone or corticosterone). Calcitonin is a naturally occurring hormone involved in calcium regulation and bone metabolism. In women over 5 years after menopause, calcitonin slows bone loss, increases spinal density, and reduces Fracture related pain. Calcitonin reduces the risk of spinal fractures. Calcitonin can be used in the form of an injection (50-100 IU per day) or a nasal spray (200 IU per day). Estrogen Therapy (ET) / Hormone Therapy (HT) is approved for the prevention of osteoporosis. ET has been shown to reduce bone loss, increase bone density in the spine and hip, and reduce the risk of hip and spine fractures in postmenopausal women. ET is most often administered in the form of a pill or dermal patch that delivers a low dose of about 0.3 mg per day or about 0.625 mg per day and is effective even at the beginning of age 70. When estrogen is administered alone, it increases the risk of women developing endometrial cancer (endometrial cancer). To eliminate this risk, health care workers prescribe hormone lutein (hormone replacement therapy or HT) in combination with estrogen for women with intact uterus. ET/HT alleviates menopausal symptoms and has been shown to have a beneficial effect on bone health. Side effects can include vaginal bleeding, breast tenderness, mood disorders, and gallbladder disease. Raloxifene, 60 mg per day, is approved for the prevention and treatment of postmenopausal osteoporosis. It is derived from a class of drugs known as selective estrogen receptor modulators (SERMs) that have been developed to provide the beneficial effects of estrogen in the absence of potential defects in estrogen. Raloxifene increases bone mass and reduces the risk of spinal fractures. There is no data available to demonstrate that raloxifene reduces the risk of hip fractures and other non-spine fractures. One form of parathyroid hormone, teriparatide, is approved for the treatment of osteoporosis in menopausal women and men at high risk of fracture. This drug stimulates new bone formation and significantly increases bone mineral density. In women after menopause, fracture reductions in the spine, hips, feet, ribs, and wrists have been documented. In men, fracture reduction in the spine has been documented, but the assessment of fracture reduction at other sites is not sufficient. Teriparatide is administered by itself in the form of a daily injection for up to 24 months.

7. Pharmaceutical composition

In certain embodiments, an activin-ActRIIa antagonist of the invention (e.g., an ActRIIa polypeptide) is formulated with a pharmaceutically acceptable carrier. For example, the ActRIIa polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds can be formulated for administration in any convenient manner for use in human or veterinary medicine.

In certain embodiments, the methods of treatment of the invention comprise administering the composition systemically or topically in the form of an implant or device. When administered, the therapeutic compositions useful in the present invention are in a pyrogen-free, physiologically acceptable form. Therapeutic agents other than ActRIIa antagonists, which may also be included in the compositions as described above, may be administered simultaneously or sequentially with the subject compounds (e.g., ActRIIa polypeptides) in the methods of the invention.

Typically, an ActRIIa antagonist will be administered parenterally and especially intravenously or subcutaneously. A pharmaceutical composition suitable for parenteral administration may comprise one or more ActRIIa polypeptides together with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or may be administered before use. A combination of sterile powders of sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostatic agents, solutes, or suspensions, which promote the formulation and the blood of the intended recipient, or suspensions Or a thickener. Examples of suitable aqueous carriers and non-aqueous vehicles which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, Vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Can The proper fluidity is maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Additionally, the composition can be encapsulated or injected for delivery to a target tissue site, such as bone. In certain embodiments, the compositions of the present invention can include the ability to deliver one or more therapeutic compounds (eg, an ActRIIa polypeptide) to a target tissue site (eg, bone), provide a structure that allows tissue to develop, and is optimally resorbable to The matrix in the body. For example, the matrix can provide a slow release of the ActRIIa polypeptide. The matrices can be formed from materials currently used in other implantable medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interfacial properties. The particular application of the subject composition will define the appropriate formulation. The potential matrix for the composition can be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyanhydride. Other potential materials are biodegradable and biologically well defined, such as collagen or dermal collagen. Other matrices contain pure protein or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminate or other ceramics. The matrix may comprise a combination of materials of any of the types mentioned above, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be altered in composition (such as calcium-aluminate-phosphate) and processing to alter pore size, particle size, particle shape, and biodegradability.

In certain embodiments, the methods of the invention may be administered orally, for example, in capsules, cachets, pills, troches, buccal formulations (using flavoring bases) , usually in the form of sucrose and acacia or tragacanth, in the form of powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, Or as a tanning agent or syrup, or as a tablet (using an inert base such as gelatin and glycerin, or sucrose and gum arabic) and/or as a mouthwash and the like, each containing a predetermined amount of the agent as an active ingredient. The medicament may also be administered in the form of a bolus, tincture or paste.

In a solid dosage form (capsules, lozenges, pills, dragees, powders, granules and the like) for oral administration, one or more therapeutic compounds of the invention may be administered with one or more pharmaceutically acceptable carriers a compound (such as sodium citrate or dicalcium phosphate) and/or any of the following: (1) a filler or supplement such as starch, lactose, sucrose, glucose, mannitol and/or citric acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar , calcium carbonate, potato or tapioca starch, alginic acid, certain citrates and sodium carbonate; (5) solution blockers, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents , such as cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene a diol, sodium lauryl sulfate, and mixtures thereof; and (10) a colorant. In the case of capsules, lozenges and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed in soft and hard-filled gelatin capsules using such excipients as lactose and high molecular weight polyethylene glycols and the like. Filler.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzene Benzyl formate, propylene glycol, 1,3-butanediol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and dehydration Fatty acid esters of sorbitol; and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

In addition to the active compound, the suspension may contain suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar And tragacanth and its mixture.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents including, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include an isotonic agent such as sugar, sodium chloride, and the like in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be facilitated by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

It will be appreciated that the dosage regimen will be determined by the attending physician under consideration of various factors that alter the effects of the subject compounds of the invention (e.g., ActRIIa polypeptide). Various factors include (but are not limited to): the amount of bone weight to be formed, the extent of bone loss, the location of bone damage, the condition of the damaged bone, the age, sex and diet of the patient, which can contribute to bone loss. The severity of the disease, the time of administration, and other clinical factors. Optionally, the dosage will vary with the type of substrate used in the reconstituted water and the type of compound in the composition. Addition of other known growth factors to the final composition can also affect the dosage. The process can be monitored by periodic assessment of bone growth and/or repair, such as X-rays (including DEXA), histomorphometry, and tetracycline labeling.

Experiments on primates and humans have demonstrated that the effect of ActRIIa-Fc on bone can be detected when the compound is administered at intervals and in amounts sufficient to achieve a serum concentration of about 200 ng/ml, with assimilated bone biomarkers. The half-maximum effect occurs at a dose of 0.3 mg/kg or according to the area equivalent under the curve. In humans, a serum dose of 200 ng/ml can be achieved in a single dose of 0.1 mg/kg or higher and a serum dose of 1000 ng/ml can be achieved in a single dose of 0.3 mg/kg or higher. The serum half-life of the observed molecule is between about 25 days and about 35 days, which is substantially longer than most Fc fusion proteins, and thus can be, for example, by about 0.05 per week or biweekly Administration to 0.5 mg/kg to achieve a sustained effective serum level, or a higher dose may be used at longer dosing intervals. For example, a dose of 0.1, 0.3, 0.5, 0.7, 1, 2, or 3 mg/kg, or between values, may be used according to monthly or bimonthly, and the effect on bone may be sufficiently durable Dosing is only required every 3 months, 4 months, 5 months, 6 months, 9 months, 12 months or more. A longer dosing interval is further supported by the duration of the pharmacodynamic effect, which is longer than the duration of the drug in the serum. Observing PD effects for at least 120 days in human patients

In certain embodiments, the invention also provides gene therapy for the production of an ActRIIa polypeptide in vivo. The therapy achieves its therapeutic effect by introducing an ActRIIa polynucleotide sequence into a cell or tissue having a disorder as listed above. Delivery of the ActRIIa polynucleotide sequence can be accomplished using a recombinant expression vector (such as a chimeric virus) or a colloidal dispersion system. Preferably, the targeted liposomes are used for the therapeutic delivery of the ActRIIa polynucleotide sequence.

Various viral vectors useful for gene therapy as taught herein include adenovirus, herpes virus, vaccinia or preferably RNA viruses (such as retroviruses). Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors into which a single foreign gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMusv) , murine mammary tumor virus (MuMTV) and Rous Sarcoma Virus (RSV). Many other retroviral vectors can combine multiple genes. All such vectors can transfer or combine the genes of the selectable marker such that the transduced cells can be identified and produced. Retroviral vectors can be made target specific by attachment, for example, to sugars, glycolipids, or proteins. Preferred targeting is achieved by the use of antibodies. Those skilled in the art will appreciate that a particular polynucleotide sequence can be inserted into the retroviral genome or ligated to the viral envelope to allow the target to specifically deliver a retroviral vector containing the ActRIIa polynucleotide. In a preferred embodiment, the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected with plastids encoding the retroviral structural genes gag, pol and env by conventional calcium phosphate transfection. These cells are then transfected with a vector plastid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for ActRIIa polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred gel system of the invention is a liposome. Liposomes are artificial membrane vesicles suitable for use as an in vitro and in vivo delivery vehicle. RNA, DNA, and intact virions can be encapsulated in an aqueous interior and delivered to cells in a biologically active form (see, eg, Fraley et al, Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using liposome vehicles are known in the art, see, for example, Mannino et al, Biotechniques, 6: 682, 1988. The composition of the liposome is typically a combination of phospholipids often combined with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of the liposome depend on the pH, ionic strength and the presence of divalent cations.

Examples of lipids suitable for the production of liposomes include phospholipid compounds such as phospholipid glycerol, phospholipid choline, phospholipid lysine, phospholipid oxime ethanolamine, sphingolipid, brain glycol and gangliosides. Illustrative phospholipids include lecithin, choline, dipalmitoside, choline, and distearylphosphocholine. Targeting of liposomes is also likely based, for example, on organ specificity, cell specificity, and organelle specificity and is known in the art.

Example

The present invention will now be described in detail with reference to the preferred embodiments of the invention Limit the invention.

Example 1: ActRIIa-Fc fusion protein

Applicants constructed a soluble ActRIIa fusion protein with a human ActRIIa ectodomain fused to a human or mouse Fc domain with a minimal link between the two. These constructs are called ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc is shown below as purified based on cell lines (SEQ ID NO: 7): from CHO ILGRS.ETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTALMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP VREKTISKAKGQPRIPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIa-hFc protein and the ActRIIa-mFc protein are expressed in the CHO cell line. Consider three different leader sequences: (i) bee melittin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 8)

(ii) Tissue plasminogen activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 9) (iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 10).

The selected sub form using the TPA leader and has the following amino acid sequence without the processing of: MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

This polypeptide is encoded by the following nucleic acid sequence: ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCC (SEQ ID NO: 14)

Both ActRIIa-hFc and ActRIIa-mFc are significantly responsive to recombinant performance. As shown in Figure 1, the protein is purified into a single, well-defined protein peak. N-terminal sequencing revealed a single sequence ILGRSETQE (SEQ ID NO: 11). Purification can be accomplished by a series of column chromatography steps including, for example, three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, Phenyl Sepharose Chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be accomplished by viral filtration and buffer exchange. ActRIIa-hFc protein purified to size exclusion layer The assay determined >98% and >95% purity as determined by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc show high affinity for ligands, especially activin A. GDF-11 or Activin A ("ActA") was immobilized on a Biacore CM5 wafer using a standard amine coupling procedure. The ActRIIa-hFc protein and the ActRIIa-mFc protein were loaded on the system, and the binding was measured. ActRIIa-hFc to 5 × 10 ^-12 solution of the dissociation constant (K _D) in combination with activin, and the protein binding to the K _D of GDF11 9.96 × 10 ^-9. See Figure 2. ActRIIa-mFc is similarly expressed.

The A-204 reporter gene assay was used to assess the effect of the ActRIIa-hFc protein on GDF-11 and activin A signaling. Cell line: human rhabdomyosarcoma (derived from muscle). Report conductor carrier: pGL3 (CAGA) 12 (described in Dennler et al., 1998, EMBO 17:3091-3100). See Figure 3. The CAGA12 motif is present in the TGF-beta response gene (PAI-1 gene), so the vector is generally used for factor signal transduction via Smad2 and 3.

Day 1: A-204 cells were split into 48 well plates.

Day 2: A-204 cells were transfected with 10 μg of pGL3 (CAGA) 12 or pGL3 (CAGA) 12 (10 μg) + pRLCMV (1 μg) and Fugene.

Day 3: Add factor (diluted to medium + 0.1% BSA). The inhibitor must be pre-incubated with the factor for 1 hour before being added to the cells. After 6 hours, the cells were washed with PBS and the cells were lysed.

Thereafter, luciferase assay was performed. Typically, in this assay, Activin A shows about 10 times the stimulating conductor base in the absence of any inhibitor. Due to performance and ED50 of about 2 ng/ml. GDF-11: 16-fold stimulation, ED50: approximately 1.5 ng/ml. The GDF-8 display is similar to the role of GDF-11.

As shown in Figure 4, ActRIIa-hFc and ActRIIa-mFc inhibit GDF-8 mediated signal transduction at Pimol concentration. As shown in Figure 5, three different ActRIIa-hFc formulations inhibited GDF-11 signaling at an IC50 of about 200 pM.

ActRIIa-hFc is extremely stable in pharmacokinetic studies. Rats were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein and plasma levels of protein were measured at 24, 48, 72, 144 and 168 hours. In an independent study, rats were dosed at 1 mg/kg, 10 mg/kg or 30 mg/kg. In rats, ActRIIa-hFc has a serum half-life of 11-14 days and the circulating dose of the drug is quite high after two weeks (11 μg for the initial dose of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively) /ml, 110 μg/ml or 304 μg/ml). In macaques, the plasma half-life is substantially longer than 14 days and the circulating dose of the drug is 25 μg/ml, 304 μg/ml or 1440 μg/ for the initial dose of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively. Ml. Preliminary results in humans indicate that the serum half-life line is between about 20 and 30 days.

Example 2: ActRIIa-mFc promotes bone growth in vivo

Normal female mice (BALB/c) were dosed with ActRIIa-mFc at a dose of 1 mg/kg/dose, 3 mg/kg/dose or 10 mg/kg/dose, wherein the dose was administered twice a week. The bone mineral density and bone mineral content were determined by DEXA, see Figure 6.

In BALB/c female mice, DEXA scan showed a significant increase in bone mineral density and content due to ActRIIa-mFc treatment (>20%). See Figure 7 and Figure 8.

Therefore, the antagonism of ActRIIa increases the bone density and content of normal female mice. As a next step, the effect of ActRIIa-mFc on the bone of a mouse model of osteoporosis was tested.

Andersson et al. (2001) established that ovariectomized mice suffered substantial bone loss (about 50% of trabecular bone loss 6 weeks after surgery) and that bone loss in these mice can be corrected by candidate therapeutic agents such as parathyroid hormones.

Applicants used ovariectomized (OVX) or sham-operated C57BL6 female mice 4-5 weeks of age. At 8 weeks after surgery, treatment with ActRIIa-mFc (10 mg/kg twice a week) or control (PBS) was initiated. Bone density was measured by a CT scanner.

As shown in Figure 9, after 6 weeks, untreated ovariectomized mice showed substantial loss of trabecular bone density relative to the mock control. ActRIIa-mFc treatment restored bone density to the level of sham-operated mice. At 6 and 12 weeks of treatment, ActRIIa-mFc caused a substantial increase in trabecular bone in OVX mice. See Figure 10. After 6 weeks of treatment, bone density increased by 24% relative to the PBS control. After 12 weeks, it increased by 27%.

In sham-operated mice, ActRIIa-mFc also caused a substantial increase in trabecular bone. See Figure 11. After 6 weeks and 12 weeks, the treatment produced a 35% increase relative to the control.

In another set of experiments, ovariectomized (OVX) or sham-operated mice as described above were treated with ActRIIa-mFc (10 mg/kg twice a week) or control (PBS) for 12 weeks. Similar to the results described above for ActRIIa-mFc, OVX mice receiving ActRIIa-mFc showed small as early as 4 weeks. The beam density increased by 15% and increased by 25% after 12 weeks of treatment (Figure 12). Sham-operated mice receiving ActRIIa-mFc showed a 22% increase in trabecular bone density and a 32% increase after 12 weeks of treatment, similarly as early as 4 weeks (Figure 13).

After 12 weeks of treatment with ActRIIa-mFc, systemic and in vitro femoral DEXA analysis showed increased bone density in treatment-induced ovariectomized mice and sham-operated mice (Figures 14A and 14B, respectively). These results were also supported by in vitro pQCT analysis of the middle femur, which demonstrated a significant increase in total bone density and cortical bone density after 12 weeks of treatment with ActRIIa-mFc. Controlled ovariectomized mice treated with vehicle showed comparable bone density to vehicle-treated control sham-operated mice (Figure 15). In addition to bone density, bone content increased after ActRIIa-mFC treatment. In vitro pQCT analysis of the middle femur confirmed a significant increase in total bone mass and cortical bone content after 12 weeks of treatment with ActRIIa-mFc, whereas mice treated with ovariectomy and sham-operated vehicle control showed comparable bone content (Figure 16). In vitro pQCT analysis of the middle femur also showed that mice treated with ActRIIa-mFc did not show changes in periplasmic circumference; however, ActRIIa-mFc treatment reduced the periostium circumference, indicating an increase in the inner surface of the femur The thickness of the cortex increases (Figure 17).

Mechanical testing of the femur determined that ActRIIa-mFc increased bone extra-features (maximum load, hardness, and fracture energy), which contributed to a significant increase in the intrinsic properties (extreme strength) of the bone. Ovariectomized mice treated with ActRIIa-mFc showed increased bone strength beyond the level of sham-treated vehicle-treated controls indicating complete reversal of the osteoporosis phenotype (Figure 18).

These data demonstrate that activin-ActRIIa antagonists increase bone density in normal female mice and, in addition, correct bone density in a mouse model of osteoporosis, Defects in bone content and ultimate bone strength.

In a further set of experiments, mice were ovariectomized or sham operated at week 4 and received placebo or ActRIIa-mFc (2 times/week, 10 mg/kg) at week 12 (Figure 19-24) Also known as RAP-11) it lasted for 12 weeks. Evaluate multiple bone parameters. As shown in Figure 19, ActRIIa-mFc increased the trabecular bone volume to total volume ratio (BV/TV) of OVX mice to sham-operated mice. ActRIIa-mFc also improved the trabecular architecture (Figure 20), increased cortical thickness (Figure 21) and improved bone strength (Figure 22). As shown in Figure 23, ActRIIa-mFc produced the desired effect at doses ranging from 1 mg/kg to 10 mg/kg.

Bone histomorphometry was performed at 2 week time points in sham-operated mice. The data presented in Figure 24 demonstrates that ActRIIa-mFc has a dual role: inhibiting bone resorption and promoting bone growth. Thus, ActRIIa-mFc stimulates bone growth (assimilation) and inhibits bone resorption (anti-alienation). BV = bone volume; TV = total tissue volume. BV/TV is a measure of the percentage of mineralized bone volume. ES = erosion surface; BS = bone surface. ES/BS is a measure of bone erosion and the decrease caused by RAP-011 confirms anti-resorption or anti-alienation. Ms/Bs is the mineralized surface/bone surface ratio, which is an indicator of bone growth or assimilation. Similarly, mineral junction rate (MAR) and bone formation rate/bone surface/day (BFR/BSd) indicate bone growth. Osteoblasts (Nob/BPm) and osteoclasts (Noc/BPm) were used to measure the mechanism of action.

A second bone histomorphometry experiment was performed in female C57BL/6 mice starting at 12 weeks of age. Mice were treated with 10 mg/kg ActRIIa-mFc Administration twice a week in the membrane lasted 2 weeks, 4 weeks, 8 weeks or 12 weeks. Each group of mice was sacrificed 5 days after the last administration and bones were obtained for analysis. The mice were subjected to calcein labeling 9 days and 2 days prior to the administration of painless lethality. As shown in Figure 25, the measurement shows that ActRIIa-mFc promotes bone growth and mineralization and has assimilation and anti-alienation. See, for example, BV/TV ratio, ES/BS ratio, and MS/BS ratio. Assimilation appears to persist throughout the dosing regimen, while anti-resorption appears to be shorter in mice.

Example 4: ActRIIa-mFc improves or prevents bone damage in a murine model of multiple myeloma

Patients with multiple myeloma show a bone loss disorder characterized by increased osteoclast activity and osteopenia formation by osteoblasts. The 5T2MM model of myeloma in mice is based on the use of tumor cells (5T2MM cells) from spontaneous tumor types that are formed in aged mice and that cause effects similar to those seen in human multiple myeloma patients in mice. . See, for example, Vanderkerken et al, Methods Mol Med. 2005; 113:191-205. ActRIIa-mFc was tested for its role in this model.

5T2 MM cells injected into C57B1/KaLwRij mice promoted an increase in osteoclast surface, osteolytic lesion formation, and reduced bone area. Osteopathology is associated with a decrease in the number of osteoblasts, a decrease in the surface of osteoblasts, and a decrease in mineralization.

Mice bearing 5T2MM cells were treated with ActRIIa-mFc (RAP-011) (10 mg/kg, intraperitoneally, twice a week) or vehicle for a total of 12 weeks from the 5T2MM injection time. MicroCT analysis of the proximal tibia and lumbar vertebra confirmed that the volume of the isolated bone in the 5T2MM mice was reduced by 39% and 21% (p<0.001 and p<0.01) and the number of trabeculae compared with the native mice. Reduced by 37% and 15% (p<0.01 and p<0.05). When compared with vehicle-treated mice, RAP-011 completely prevented the reduction of trabecular volume and number in the tibia induced by 5T2MM (p<0.001 and p<0.05) and the trabecular volume and number in the vertebrae. Reduction (p<0.01 and p<0.05). The bone volume of the RAP-011 treated mice was 19% higher in the tibia (p=168) and 12% higher in the vertebrae (p<0.05) when compared to the native mice. RAP-011 prevented the development of osteolytic lesions (p<0.05). This effect is illustrated in Figure 26. Although the initial assessment of data in this study failed to identify significant effects on serum paraprotein (biomarkers of multiple myeloma cells) or myeloma burden, further analysis indicated that serum paraproteins were treated in all but one Substantially reduced in animals and further indicates a substantial increase in the volume of healthy bone marrow, which indicates a decrease in myeloma cell load.

Therefore, ActRIIa-mFc can be used to reduce the effects of bone diseases caused by multiple myeloma and to treat tumor cells themselves.

Example 5: Characterization of ActRIIa-hFc protein

The ActRIIa-hFc fusion protein was expressed in CHO-DUKX B11 cells stably transfected from the pAID4 vector (SV40 ori/enhancer, CMV promoter) using the tissue plasminogen leader sequence of SEQ ID NO:9. The protein purified as described in Example 1 above has the sequence of SEQ ID NO: 7. The Fc portion is the human IgG1 Fc sequence as shown in SEQ ID NO: 7. Sialic acid analysis revealed that the protein contained a sialic acid/ActRIIa-hFc fusion protein molecule with an average of between about 1.5 moles and 2.5 moles.

This purified protein showed significantly longer serum half-life in all animals tested, including half-life of 25-32 days in human patients (see See Example 6, below). In addition, the substance exhibited by CHO cells has a higher affinity for activin B ligand than that reported for ActRIIa-hFc fusion protein expressed in human 293 cells (del Re et al, J Biol Chem. 2004 12 17th; 279(51): 53126-35). In addition, the use of the tPa leader sequence provides higher yields than other leader sequences, and unlike the ActRIIa-Fc represented by the native leader, it provides a high purity N-terminal sequence. Two major classes of ActRIIa-Fc were generated using native leader sequences, each with a different N-terminal sequence.

Example 6: Human Clinical Trial

The protein described in Example 5 was administered to a human patient in a randomized, double-blind, placebo-controlled study to conduct a study to primarily assess the safety of the protein in women after healthy menopause. 48 individuals were randomized into 6 groups to receive a single dose of ActRIIa-hFc or placebo (5 groups of active agents: 1 group of placebo). Dosage levels range from 0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kg subcutaneously (SC). All individuals performed this for 120 days. Individuals who were taking drugs that affect bone metabolism within 6 months of the study were excluded from the study participants. Safety assessments were performed on a per-group basis to determine the proportional scaling of the dose. In addition to pharmacokinetic (PK) analysis, the biological activity of ActRIIa-hFc was also assessed by measuring biochemical markers of bone formation and resorption and FSH content.

No serious adverse events were reported in this study. Adverse events (AEs) are generally mild and transient. Preliminary analysis of AE included headache, elevated experimental values, cold symptoms, vomiting, intravenous infiltration, and hematoma at the injection site.

PK analysis of ActRIIa-hFc showed linear characteristics with dose and Average half-life of 25-32 days. The area under the curve (AUC) of ActRIIa-hFc is linearly related to the dose and is substantially completely absorbed after subcutaneous administration (see Figures 27 and 28). These data indicate that the desired method of administration is subcutaneously because it provides the equivalent bioavailability of the drug and the serum half-life, while avoiding spikes in the serum concentration of the drug associated with the first few days of intravenous administration ( See Figure 28). ActRIIa-hFc causes a rapid, sustained dose-dependent increase in serum content of bone-specific alkaline phosphatase (BAP), which is a marker of assimilation of bone growth, and a C-terminal type 1 collagen tail peptide and tartrate-resistant acid phosphatase The 5b content is dose dependently reduced (which is a marker of bone resorption). Other markers such as P1NP show inconclusive results. The BAP content showed a near saturation effect at the highest drug dose, indicating that the half maximal effect on this assimilated bone biomarker can be achieved at a dose of 0.3 mg/kg (increase range to 3 mg/kg). Calculated according to the pharmacodynamic effect of the drug and AUC, the EC50 was 51,465 (days x ng/ml). See Figure 29. These bone biomarker changes lasted for about 120 days at the highest dose level tested. Consistent with activin inhibition, there was also a dose-dependent decrease in serum FSH content.

A single dose of ActRIIa-hFc administered to a woman after healthy menopause is safe and well tolerated for the range of dose levels tested. PK and prolonged pharmacodynamic effects indicate that intermittent administration will be appropriate for future studies. For example, administration based on serum half-life can be performed once a month or according to the rules every two weeks, three weeks, four weeks, five weeks, or six weeks. In addition, since the pharmacodynamic effect is extended far beyond the serum retention period of the drug, it can be administered based on the pharmacodynamic effect, which means every three months or every two. Administration of three, four, four, five, six or even twelve months can effectively produce the desired effect in the patient. This clinical trial confirmed that ActRIIa-hFc is a bone anabolic agent with biological signs of increased bone formation and reduced bone resorption in humans.

Example 7: Co-administration of ActRIIa-mFc with bisphosphonate

Bisphosphonates are a class of drugs that are widely used to treat conditions associated with low bone mineral density, including osteoporosis and cancer-related bone loss. Bisphosphonates have potent anti-resorption activity that inhibits osteoclasts. It may be due to osteoclasts being required for bone rupture and bone growth, so bisphosphonates appear to reduce the effects of parathyroid hormone (PTH) (only one of the known assimilated bone growth agents) (Black et al., N Engl) J Med. September 25, 2003; 349(13): 1207-15; Samadfam et al., Endocrinology. June 2007; 148(6): 2778-87).

To test the utility of ActRIIa-Fc treatment in patients who have previously received or are being concomitantly receiving bisphosphonates or other anti-reabsorption therapies, mice are combined with ActRIIa-mFc and zoledronate (a bisphosphine) The acid salt compound was tested. Treatment of 12-week-old C57BL/6N mice as follows: Group 1 PBS group 2 ActRIIa-mFc (RAP-011) (10 mg/kg), twice a week (with group 3 and group 4) group 3 zoledronic acid (Zoledronic Acid) (ZOL), 4 ZOL (1 dose) in a single dose (20 mg/kg) group, ActRIIa-mFc (RAP-011) (1 mg/g) twice daily after 3 days Group 5 ZOL (1 dose), 3 days after ActRIIa-mFc (RAP-011) (10 mg/kg) twice a week and 3 weeks and 8 weeks after treatment by DEXA scan (PIXI) BMD.

As shown in Figure 30, total BMD was significantly increased in all treatment groups, with the combination of ZOL and ActRIIa-mFc producing the greatest effect. These results indicate that the ActRIIa-Fc protein can be used to increase bone density even in patients who have received bisphosphonate therapy.

Example 8: ActRIIa-Fc improves or prevents bone loss caused by breast cancer metastasis

It is estimated that between 65% and 75% of breast cancers metastasize to the bone, which causes substantial damage to the bone structure, increases the risk of fracture and causes pain and other side effects. We tested the role of ActRIIa-Fc in a mouse model of breast cancer that has metastasized to bone.

Culture of human breast cancer cell line subline of MDA-MB-231 (pure lines 2287) and in vitro to cells collected / ml of the density of 5 × 10 ⁶ cells. MDA-MB-231 is a highly competent cell line that is inoculated into bone and causes bone damage similar to bone damage caused by bone metastasis. On day 0 of the study, 10 ml of cells were injected into the tibia of a 6-week-old female athymic nude mouse. On day 10 of the study, mice received ActRIIa-mFc (10 mg/kg/twice/subcutaneously) (n=8) or PBS vehicle (n=7). Disease progression was assessed by dual energy x-ray absorptiometry (PIXIMus) at weekly intervals. Mice were treated with ActRIIa-mFc for 4 weeks and then sacrificed, and tibia (tumor injected and tumor free) were collected from each animal. The tibia is then processed and ready for microCT and histological analysis.

MDA-MB-231 cells were injected intra-tibia into athymic nude mice to promote the development of osteolytic lesions in the injected tibia compared to the contralateral leg. MicroCT analysis of the proximal tibia confirmed a 62% reduction in the volume of the stromate bone loaded with MDA-MB-231 tibia compared to the tumor-free tibia of mice treated with PBS vehicle. Treatment with ActRIIa-mFc resulted in a 70% or 147% increase in native tibia or loaded tumor tibia, respectively (P < 0.01 for both). The tumor-bearing tibia of mice treated with ActRIIa-mFc had a similarly low bone mineral density (p=0.39) compared to the native tibia of VEH-treated mice.

Therefore, ActRIIa-mFc can eliminate bone damage associated with the presence of breast tumor cells in the bone.

Example 9: Replacement of ActRIIa-Fc protein

The alternative construct can lack the C-terminal tail of the extracellular domain of ActRIIa (the last 15 amino acids). The sequence of the construct presented below (Fc portion underlined) (SEQ ID NO: 12) : ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMLKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Incorporated by reference All publications and patents referred to herein are hereby incorporated by reference in their entirety in their entirety in their entirety in the particularties

The above description is illustrative and not limiting, although specific specific examples of the subject matter have been discussed. Many variations will become apparent to those skilled in the art after reviewing this specification and the following claims. The full scope of the invention should be determined by reference to the appended claims and the claims

Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells. The protein is purified into a single, well-defined peak.

FIG 2 shows as a BiaCore ^TM assay measuring the amount of binding ActRIIa-hFc to activin and GDF-11's.

Figure 3 shows an illustration of the A-204 reporter gene assay. The figure shows the conductor carrier: pGL3 (CAGA) 12 (described in Dennler et al., 1998, EMBO 17:3091-3100). The CAGA12 motif is present in the TGF-β response gene (PAI-1 gene), so the vector is generally used for factor signal transduction via Smad 2 and 3.

Figure 4 shows the effect of ActRIIa-hFc (diamonds) and ActRIIa-mFc (squares) on GDF-8 signal transduction in the A-204 reporter gene assay. Both proteins showed substantial inhibition of GDF-8 mediated signal transduction at Pimol concentration.

Figure 5 shows the effect of three different ActRIIa-hFc preparations on GDF-11 signal transduction in the A-204 reporter gene assay.

Figure 6 shows an example of a DEXA image of a control treated and ActRIIa-mFc treated BALB/c mouse before the 12 week treatment period (upper panel) and after the 12 week treatment period (lower panel). A lighter shade indicates an increase in bone density.

Figure 7 shows quantification of the effect of ActRIIa-mFc on bone mineral density of BALB/c mice over a 12 week period. Treatment was control (diamond), 2 mg/kg dose of ActRIIa-mFc (square), 6 mg/kg dose of ActRIIa-mFc (triangle) and 10 mg/kg dose of ActRIIa-mFc (circle).

Figure 8 shows quantification of the effect of ActRIIa-mFc on bone mineral content of BALB/c mice over a 12 week period. Treatment was control (diamond), 2 mg/kg dose of ActRIIa-mFc (square), 6 mg/kg dose of ActRIIa-mFc (triangle) and 10 mg/kg dose of ActRIIa-mFc (circle).

Figure 9 shows quantification of the effect of ActRIIa-mFc on bone mineral density of trabecular bone of ovariectomized (OVX) or sham-operated (SHAM) C57BL6 mice after a 6-week period. Treatment was control (PBS) or a dose of 10 mg/kg ActRIIa-mFc (ActRIIa).

Figure 10 shows quantification of the effect of ActRIIa-mFc on trabecular bone of ovariectomized (OVX) C57BL6 mice over a 12 week period. Treatment was control (PBS; light bars) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa; dark bars).

Figure 11 shows quantification of the effect of ActRIIa-mFc on trabecular bone of sham-operated C57BL6 mice after a 6 or 12 week treatment period. Treatment is right Photograph (PBS; light bar) or 10 mg/kg dose of ActRIIa-mFc (ActRIIa; dark bars).

Figure 12 shows the results of pQCT analysis of bone mineral density in 12-week treated ovariectomized mice. Treatment was control (PBS; light bars) or ActRIIa-mFc (dark bars). Y-axis: mg/ccm.

Figure 13 depicts the results of a pQCT analysis of bone mineral density in 12-week treated sham-operated mice. Treatment was control (PBS; light bars) or ActRIIa-mFc (dark bars). Y-axis: mg/ccm.

Figures 14A and 14B show whole body DEXA analysis (A) and in vitro analysis of femur (B) after 12 weeks of treatment. The illuminating area depicts the area of high bone density.

Figure 15 shows in vitro pQCT analysis of the mid-femoral segment after 12 weeks of treatment. Treatment was vehicle control (PBS, dark bars) and ActRIIa-mFc (light bars). The four bars on the left show total bone density, while the four bars on the right show cortical bone density. The first of the four bars in each group represents data from ovariectomized mice, while the second pair of bars represents data from sham-operated mice.

Figure 16 shows in vitro pQCT analysis and bone mineral content of the mid-femoral segment after 12 weeks of treatment. Treatment is vehicle control (PBS, dark bars) or ActRIIa-mFc (light bars). The four bars on the left show total bone content, while the four bars on the right show cortical bone content. The first of the four bars in each group represents data from ovariectomized mice, while the second pair of bars represents data from sham-operated mice.

Figure 17 shows an in vitro pQCT analysis of the mid-femur and femoral cortical thickness. Treatment was control (PBS, dark bars) and ActRIIa-mFc (light bars). The four bars on the left show the circumference of the endosteal, while the four on the right show the periosteal circumference. The first of the four bars in each group represents data from ovariectomized mice, while the second pair of bars represents data from sham-operated mice.

Figure 18 depicts the results of a mechanical test of the femur after 12 weeks of treatment. Treatment was control (PBS, dark bars) and ActRIIa-mFc (light bars). The two bars on the left represent data from ovariectomized mice, while the last two bars represent data from sham-operated mice.

Figure 19 shows the effect of ActrIIa-mFc on trabecular bone volume.

Figure 20 shows the effect of ActrIIa-mFc on the trabecular architecture in the distal femur.

Figure 21 shows the effect of ActrIIa-mFc on cortical bone.

Figure 22 shows the effect of ActrIIa-mFc on the mechanical strength of bone.

Figure 23 shows the effect of different doses of ActRIIa-mFc on bone characteristics at three different doses.

Figure 24 shows bone histomorphometry indicating that ActRIIa-mFc has dual activities of assimilation and anti-resorption.

Figure 25 shows other histomorphometric data.

Figure 26 shows images of mouse femurs from native mice and tumor-bearing mice, and the effect of ActRIIa-mFc treatment on bone morphology in a multiple myeloma model. Mice loaded with multiple myeloma (5T2) showed significant potholes and degeneration of bone relative to normal mice (native). Treatment with ActRIIa-mFc abolished this effect.

Figure 27 shows the results from the human clinical trial described in Example 6, wherein the ActRIIa-hFc is intravenous (IV) or transdermally Under (SC) administration, the area under the curve (AUC) was linearly related to the dose of ActRIIa-hFc administered.

Figure 28 shows a comparison of SerRIIa-hFc serum levels in patients administered intravenously or subcutaneously.

Figure 29 shows bone alkaline phosphatase (BAP) levels in response to different ActRIIa-hFc dose levels. BAP is a marker of assimilation of bone growth.

Figure 30 shows the synergistic effect of ActRIIa-mFc (RAP-011) and bisphosphonate agent (zoledronate) in mice.

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Claims (63)

A pharmaceutical composition comprising an ActRIIa-Fc fusion protein expressed from a CHO cell, wherein the ActRIIa-Fc fusion protein is an amino acid comprising SEQ ID NO: 7 each joined by a disulfide bond A dimer formed from a polypeptide of at least 95% identical amino acid sequence, wherein the N-terminus of each of the polypeptides is ILGRSETQE, and wherein the dimer comprises 3 or more sialic acid moieties. The pharmaceutical composition of claim 1, wherein the dimer comprises from 3 to 5 sialic acid moieties. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein is formed from two polypeptides each comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 7. Dimer. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising the amino acid sequence of SEQ ID NO: 7. The pharmaceutical composition of claim 1, wherein the dimer comprises 4 sialic acid moieties. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein has an average serum half-life of 25 to 32 days in a normal and healthy person and is equivalent when administered intravenously or subcutaneously. Biological availability. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for subcutaneous administration. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein has a purity of at least 90% relative to other protein components. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein binds to activin. The pharmaceutical composition of claim 9, wherein the ActRIIa-Fc fusion protein binds to activin A. The pharmaceutical composition of claim 1, wherein the ActRIIa-Fc fusion protein binds to GDF11. Use of an ActRIIa-Fc fusion protein for the manufacture of a medicament for treating or preventing cancer-related bone loss in a human patient, wherein the ActRIIa-Fc fusion protein comprises an amino acid sequence comprising the same as SEQ ID NO: a polypeptide of at least 95% identical amino acid sequence, wherein the N-terminus of the polypeptide is ILGRSETQE, wherein the ActRIIa-Fc fusion protein binds to activin or GDF11, and wherein the cancer-associated bone loss is associated with multiple myeloma Bone loss or bone loss associated with breast cancer. The use of claim 12, wherein the ActRIIa-Fc fusion protein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7. The use according to claim 12, wherein the ActRIIa-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 7. The use of claim 12, wherein the ActRIIa-Fc fusion protein binds to activin. The use of the ActRIIa-Fc as claimed in claim 15 The fusion protein binds to activin A. The use according to claim 12, wherein the ActRIIa-Fc fusion protein binds to GDF11. The scope of the patent application 242,800 of 12, wherein the ActRIIa-Fc fusion protein has one or more of the following features of: i to K _D and ActRIIa ligand binding of at least 10 ^-7 M; and (ii) inhibition of cell. ActRIIa signal transduction. The use according to claim 12, wherein the ActRIIa-Fc fusion protein comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, and methods An amino acid, an acetamino acid, an amino acid bound to biotin, an amino acid bound to a lipid moiety, and an amino acid combined with an organic derivatizing agent. The use of claim 12, wherein the dimer comprises three or more sialic acid moieties. The use according to claim 12, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising an amino acid sequence at least 99% identical to SEQ ID NO: 7. The use according to claim 12, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising the amino acid sequence of SEQ ID NO: 7. The use of claim 12, wherein the ActRIIa-Fc fusion protein comprises from 3 to 5 sialic acid moieties. The use of claim 12, wherein the pharmaceutical product increases the skeletal muscle mass of the patient by less than 10%. The use of claim 12, wherein the medicament is for administering a serum concentration of at least 200 ng/mL of the ActRIIa-Fc fusion protein in the patient. The use of claim 25, wherein the pharmaceutical is administered to achieve a serum concentration of at least 1000 ng/mL of ActRIIa-Fc fusion protein in the patient. The use of claim 12, wherein the ActRIIa-Fc fusion protein has a serum half-life between 15 and 40 days in a normal, healthy person. The use of claim 22, wherein the pharmaceutical product is for administering the patient at a frequency no higher than once a week. The use of claim 22, wherein the pharmaceutical product is for administering the patient at a frequency no higher than once a month. The use of claim 22, wherein the pharmaceutical product is for administering the patient at a frequency not higher than once every three months. The use of any one of clauses 12 to 30, wherein the patient is undergoing skeletal antiresorption therapy or has received skeletal antiresorption therapy within one year prior to administration of the medicinal product. The use of claim 31, wherein the anti-resorptive agent is selected from the group consisting of a bisphosphonate agent, a RANK ligand antagonist, and an osteoprotegrin antagonist. The use of any one of clauses 12 to 30, wherein the pharmaceutical is for administration to a human patient in combination with a radiation therapy, a cytotoxic agent, or a chemotherapeutic agent. The use of any one of clauses 12 to 30, wherein the patient has multiple myeloma. Use of an ActRIIa-Fc fusion protein for the manufacture of a medicament for treating or preventing multiple myeloma in a human patient, wherein the ActRIIa-Fc fusion protein comprises an amino acid sequence comprising the same as SEQ ID NO: A polypeptide of at least 95% identical amino acid sequence, and wherein the ActRIIa-Fc fusion protein binds to activin or GDF11. The use of the 35th aspect of the invention, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 3. The use according to claim 35, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. The use of the 35th aspect of the invention, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2. The use of claim 35, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 2. The use of the 35th aspect of the patent application, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. The use of the 35th aspect of the patent application, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 7. For example, the application of patent scope 35, where ActRIIa-Fc The protein comprises a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7. The use according to claim 35, wherein the ActRIIa-Fc fusion protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 7. The use of claim 35, wherein the ActRIIa-Fc fusion protein binds to activin. The use according to claim 44, wherein the ActRIIa-Fc fusion protein binds to activin A. The use of claim 35, wherein the ActRIIa-Fc fusion protein binds to GDF11. The scope of the patent application of the use of 35, wherein the ActRIIa-Fc fusion protein has one or more of the following features of: i to K _D and ActRIIa ligand binding of at least 10 ^-7 M; and (ii) inhibition of cell. ActRIIa signal transduction. The use of claim 35, wherein the ActRIIa-Fc fusion protein comprises one or more modified amino acid residues selected from the group consisting of glycosylated amino acids, PEGylated amino acids, and methods An amino acid, an acetamino acid, an amino acid bound to biotin, an amino acid bound to a lipid moiety, and an amino acid combined with an organic derivatizing agent. The use according to claim 35, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2. And wherein the dimer comprises three or more sialic acid moieties. The use of the ActRIIa-Fc as claimed in claim 49 The fusion protein is a dimer formed from two polypeptides each comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 2. The use according to claim 49, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising the amino acid sequence of SEQ ID NO: 2. The use according to claim 49, wherein the ActRIIa-Fc fusion protein is dimerized from two polypeptides each comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 7. body. The use according to claim 49, wherein the ActRIIa-Fc fusion protein is dimerized from two polypeptides each comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 7. body. The use according to claim 49, wherein the ActRIIa-Fc fusion protein is a dimer formed from two polypeptides each comprising the amino acid sequence of SEQ ID NO: 7. The use of claim 49, wherein the ActRIIa-Fc fusion protein comprises from 3 to 5 sialic acid moieties. The use of claim 35, wherein the pharmaceutical product increases the skeletal muscle mass of the patient by less than 10%. The use of claim 35, wherein the pharmaceutical product is for administration to a serum concentration of at least 1000 ng/mL of ActRIIa-Fc fusion protein in the patient. The use of claim 35, wherein the ActRIIa-Fc fusion protein has a serum half-life between 15 and 40 days in a normal, healthy person. The use of claim 54, wherein the pharmaceutical product is for administering the patient at a frequency no higher than once a week. The use of claim 54 wherein the pharmaceutical product is for administration to the patient at a frequency no greater than once a month. The use of claim 54 wherein the pharmaceutical product is for administration to the patient at a frequency not higher than once every three months. The use of claim 35, wherein the N-terminus of the polypeptide is ILGRSETQE. For example, the use of the scope of claim 35, wherein the pharmaceutical product reduces the burden of myeloma tumors in the patient.